Human Biochem Musc
Human Biochem Musc
Human Biochem Musc
Michael Palmer, MD
Department of Chemistry
University of Waterloo
Waterloo, ON N2L 3G1
Canada
mpalmer @ uwaterloo.ca
This version is from Friday 10th April, 2015. The latest electronic version of these notes
and accompanying slides is available for free at
watcut.uwaterloo.ca/webnotes/Metabolism/.
Copyright© Michael Palmer, 2014. All rights reserved (see section 20.3 for details). Cover design by
Jana Rade (impactstudiosonline.com). The cover illustration is a model of a glycogen molecule.
Preface
These lecture notes, and the accompanying slides, are intended for teaching human
metabolism at the undergraduate level. They aim to give a big picture view of the
field that takes into account physiological and some clinical aspects. They also
strive to be up to date. Some biochemistry texts present all the latest and greatest
enzyme crystal structures, but seem stuck in the 1960s when it comes to updating
metabolic pathways. It is likely that these notes are not completely free from
this disease either, and I therefore welcome any corrections and suggestions for
improvement.
The notes and the slides are meant to be used in conjunction; the notes mostly
present the slides in sequence, augmenting each with explanatory text. Keeping
the figures and the corresponding text closely together may not always look as
polished as a conventional book layout, but it makes on-screen reading easier. This
structure also encourages me to stay on topic and to advance the plot with each
successive slide. Finally, it makes it easier to recapitulate or anticipate the content
of a lecture; this goes for both the students and the lecturer.
Another conscious choice is to make these materials freely available. The text
is entirely my own work. So is the majority of the images; all exceptions are listed
in chapter 20. Regarding those images, I would like to thank all individuals and
institutions who gave me permission to reuse them in these notes. In particular, I
thank Katharina Glatz of Basel University for permission to use multiple histologi-
cal pictures from her excellent website pathorama.ch. You are free and welcome to
use these notes and slides for self-study or for classroom teaching. Some restric-
tions apply concerning the copying and redistribution of these materials; refer to
the copyright notice in chapter 20 for details. I hope that, in return, you will bring
to my attention any errors or shortcomings that you may notice.
I would also like to thank my students, who have helped to improve these
notes by challenging and questioning me about the material and its presentation.
In particular, Stefanie Malatesta and Julia Plakhotnik have helped substantially in
this manner.
These notes were put together with a collection of excellent free software
tools. Apart from the well-known tools LATEX, Pymol, and Gnuplot, I would like to
highlight the outstanding chemfig LATEX package written by Christian Tellechea
that was used to draw all of the chemical structures and reaction mechanisms.
iii
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Chapter 1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Motivation: Why would you study metabolism? . . . . . . . . . . . . . . 1
1.2 Significance of metabolism in medicine . . . . . . . . . . . . . . . . . . . . 1
1.3 Catabolic and anabolic reactions . . . . . . . . . . . . . . . . . . . . . . . . 2
1.4 Diversity of metabolism: pathways in plants and bacteria . . . . . . . . 3
1.5 Types of foodstuffs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.6 The digestive system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.7 Answers to practice questions. . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 2 Refresher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.1 Preliminary note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2 How enzymes work: active sites and catalytic mechanisms . . . . . . . 17
2.3 Energetics of enzyme-catalyzed reactions . . . . . . . . . . . . . . . . . . 20
2.4 The role of ATP in enzyme-catalyzed reactions . . . . . . . . . . . . . . . 22
2.5 Regulation of enzyme activity . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.6 Answers to practice questions. . . . . . . . . . . . . . . . . . . . . . . . . . 29
Chapter 3 Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1 Overview of glucose metabolism . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2 Reactions in glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3 Mechanisms of enzyme catalysis in glycolysis . . . . . . . . . . . . . . . 34
3.4 Glycolysis under aerobic and anaerobic conditions . . . . . . . . . . . . 40
3.5 Transport and utilization of glucose in the liver and in other organs. 42
3.6 Answers to practice questions. . . . . . . . . . . . . . . . . . . . . . . . . . 44
v
Contents
vi
Contents
vii
Contents
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
viii
Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
ix
Chapter 1
Introduction
Long answer: You have a generous and warm character, and you have spent too
much time in front of the family TV set. You are therefore determined to become
a famous doctor and save many, many lives every hour of the day, without asking
anything in compensation but the admiring gazes of the populace, and may be a
Rolls Royce. Metabolism is an ever so tiny part of the vast knowledge you have
set out to master in order to fulfill your destiny.
Wrong? Alternate long answer: You have the inquisitive mind of a Sherlock
Holmes and the financial savvy of a Howard Hughes, and you have determined
that soaking medical doctors for damages is the best road to wealth and fame. Un-
derstanding the biochemical basis of medicine will help you to stun your audiences
in court and grind the defendants and their counsels into the dust.
Wrong again? Then try the short answer: You want to pass your exam.
1
2 1 Introduction
Complex
biomolecules
NADP+
NADPH + H+
ADP + Pi O2
ATP
Notes: The metabolism of animals and humans can be divided into catabolic re-
actions (blue arrows) and anabolic ones (green). The word “catabolic” means
the same as “degradative,” but it is Greek and therefore sounds a whole lot more
erudite and scholarly. A large share of the substrates broken down in catabolism
are used for producing ATP, the “electric energy” of the cell. Just as electricity
can be used to drive just about any household job, ATP is used for almost every
energy-requiring task in cell biology. Because of its key role in the life of the cell,
we will devote a good deal of space—chapters 3–6—to the metabolic pathways that
allow the cell to regenerate ATP.
The word “anabolic” might be translated as “constructive.” Anabolic pathways
are the opposite of catabolic ones, that is, they create new biomolecules. They
produce small molecules and building blocks that are not sufficiently available
1.4 Diversity of metabolism: pathways in plants and bacteria 3
Pathway Organisms
Notes: While the scope of this text is mostly restricted to human metabolism, it is
useful to take a brief look beyond these confines. There are several mainstream
metabolic pathways that occur in all classes of living organisms. A good example is
glycolysis, the main pathway of glucose degradation, which is found all the way up
from Escherichia coli to Homo sapiens. On the other hand, some of the metabolic
processes in plants or in distinct groups of microbes are quite different from those
found in man or animals.
Photosynthesis enables plants to create glucose—and from it, the carbon
skeletons of all their other metabolites—from nothing but CO2 and water. The
same is true of blue-green algae or cyanobacteria.1 Organisms incapable of photo-
synthesis are heterotrophic, which means that they must feed on other organisms.
In contrast, photosynthesis makes organisms autotrophic, that is, capable of feed-
ing themselves.2 Note, however, that plant life also depends on pathways other
than photosynthesis. An example is the degradation of starch, which is stored in
large amounts in plant seeds such as wheat and rice as well as in bulbs such as
potatoes. The pathways of starch utilization employed by plants are analogous to
those found in animals.
1
Indeed, cyanobacteria are the prokaryotic precursors of chloroplasts, the photosynthetic or-
ganelles of plants. Like mitochondria, chloroplasts are endosymbionts of prokaryotic origin and still
retain their own genomes and ribosomes.
2
Carnivorous plants could be considered both autotrophs and heterotrophs. It seems, however,
that plants evolved this lifestyle mostly to secure a supply of organic nitrogen rather than of carbon.
4 1 Introduction
Nitrogen fixation, that is, the reduction of atmospheric nitrogen (N2 ) to ammo-
nia (NH3 ), is performed by the bacterium Sinorhizobium meliloti and related soil
bacteria. All other living organisms require nitrogen in already reduced form, and
therefore depend on these bacteria. The word Rhizobium—the former first name
of this bacterium—means “living on roots”. Sinorhizobium meliloti thrives on the
roots of plants, which take up the surplus ammonia supplied by the bacteria and
utilize it for synthesizing their own amino acids; the plants, in turn, provide the
bacteria with a nutrient-rich environment. This symbiotic process is common with
legumes such as alfalfa, soy beans, and peas. Including these plants in crop rota-
tion schemes helps to keep the soil supplied with reduced nitrogen. Alternatively,
the nitrogen fixation bottleneck can be bypassed altogether by supplying reduced
nitrogen with chemical fertilizers. 3
Some archaebacteria, which live in exotic environments such as submarine
volcanic hot springs, have developed correspondingly exotic metabolic pathways.
For example, some of these organisms are capable of extracting energy from the
oxidation of iron or the reduction of sulfur.
While all these pathways are certainly very interesting, we will not consider
them any further in these notes. Instead, we will confine the discussion to the
major metabolic pathways that occur in the human body. We will also relate these
pathways to human health and disease, and to some of the therapeutic strategies
that have been developed for metabolic diseases.
In the remainder of this chapter, we will start with a broad overview of food-
stuffs and their digestion and uptake in the intestinal organs. This will provide
some important context for the detailed discussion of metabolic pathways in the
subsequent chapters.
1.2 In the metabolism of heterotrophs like Homo sapiens, what are the ultimate sources
of organic carbon and of reduced nitrogen?
• carbohydrates
• protein
• fat
• nucleic acids
3
Until the early 20th century, the only practical source of nitrogen fertilizer was guano, which
is accumulated, dried bird poop. It is found in large deposits on cliffs and islands off the South
American west coast, and clippers ferrying the precious stuff used to go back and forth between
Chile or Peru and Europe or North America.
In 1909, Haber and Bosch devised a synthetic method for producing nitrogen fertilizer, as well as
nitrogen-based explosives. In this process, the reaction N2 + 3 H2 2 NH3 is induced by brute force:
a mixture of the two gases is compressed to very high pressures and heated to high temperature
in the presence of a metal catalyst. The method has been in use ever since for producing nitrogen
fertilizer. Among all the great inventions that have propelled the growth of the world population,
the Haber-Bosch process likely is the single most important one.
1.5 Types of foodstuffs 5
Notes: The three major categories of foodstuffs relevant to human metabolism are
named on every box of cereal or cup of yogurt; and in case you do not remember
them, I suggest you run out right now to buy some such scholarly piece of grocery;
dollar for dollar, its educational value might exceed that of your attendance of this
class.
The fourth item in the list above, nucleic acids, could as well have been sub-
sumed under carbohydrates, since only the ribose and deoxyribose contained in
them have significant nutrient value. The pathways that would allow the reuse of
the bases for nucleotide and nucleic acid synthesis exist in principle, but experi-
mental studies indicate that ingested bases are mostly degraded and excreted (see
section 16.4).
ADP + Pi
ATP
CO2 + H2 O
Notes: In the first stage of their utilization, all foodstuffs are split into their build-
ing blocks; this happens mostly during digestion in the small intestine. After the
building blocks—mainly glucose, amino acids, and fatty acids—have been taken up
and distributed through the blood stream, complete breakdown to CO2 and H2 O
proceeds intracellularly via pyruvate and acetyl-CoA, which function as central
hubs of foodstuff utilization.
Glucose can transiently be stored in polymeric form as glycogen, which evens
out the peaks and valleys of glucose supply during the day. If required, additional
glucose can be produced via gluconeogenesis from amino acids whose degradation
yields pyruvate.
When other forms of substrate carbon are in short supply, fatty acids—either
taken up with the food or released from fat tissue—can be converted via acetyl-
CoA to ketone bodies, which represent a more water-soluble transport form of
carbon than the fatty acids themselves.
This slide is of course simplified and contains several approximations. For
example, some carbohydrates do not directly yield glucose upon depolymerization;
these may then be converted to glucose through dedicated adapter pathways. Sim-
ilarly, the breakdown of some amino acids does not yield pyruvate or acetyl-CoA
6 1 Introduction
but instead produces citric cycle intermediates. Like pyruvate, these intermediates
can also be converted to glucose if needed.
1.3 Name the three major products of foodstuff digestion in the small intestine, and the
two central hubs of cellular foodstuff utilization.
Pancreas
Bile duct
Bile bladder
Pancreatic duct
Notes: The digestive system contains the intestinal hollow organs, that is, the
esophagus, stomach, small and large intestine. In addition, it also comprises the
pancreas and the liver, both of which arise through budding and outgrowth from
the primordial intestine during embryonic development.
Organ Function
Notes: The mucous membrane of the stomach produces gastric acid, HCl, which
denatures proteins and kills microbes contained in the food. The pancreas supplies
most of the digestive enzymes, whereas the liver provides bile acids, which are
1.6 The digestive system 7
essential for the solubilization of fat. The bile and the pancreatic juice also contain
large amounts of sodium bicarbonate, which neutralizes the gastric acid; the milieu
inside the small intestine is slightly alkaline.4
The digestive enzymes secreted by the pancreas into the gut do most of the
work involved in digestion of ingested foodstuffs. Therefore, depolymerization of
foodstuff macromolecules occurs extracellularly.5
1.4 Name the digestive organs and summarize their functions.
Systemic circulation
Abdominal arteries
Liver vein
Liver artery
Portal vein
Notes: Upon uptake, most solutes will be exported on the other side of the mu-
cosal cells and then find themselves in the blood stream. A peculiarity of the
intestines is that all blood drained from them is first passed through the liver
before being released into the general circulation. This serves a twofold purpose:
1. It gives the liver a chance to take excess amounts of substrates—glucose,
amino acids—out of circulation and to store and process them. This serves to
4
The bile duct and the pancreatic duct join the duodenum, that is, the uppermost part of the
small intestine, at the same site (termed the papilla duodeni major). Bile stones traveling down the
bile duct may get stuck at this orifice and obstruct both secretory ducts. On top of bile colics, this
may then result in acute pancreatitis, in which the backed-up pancreatic enzymes start digesting the
pancreas itself. This is both exceedingly painful and a major, acutely life-threatening calamity.
5
Extracellular digestion is employed by most organisms. Even bacteria secrete digestive enzymes
and take up substrates only at the stage of the monomeric breakdown products. Why is that so?
An obvious answer is that there are no transport mechanisms for the uptake of macromolecules
across the cell wall. While that is true, there is a deeper reason—taking up macromolecules in
a non-specific way would open the door for all kinds of viruses and Trojan horses. Extracellular
digestion constitutes a firewall that excludes hazardous macromolecules.
Exceptions to the rule above are amoebas, which ingest not only macromolecules but even whole
bacteria. However, the ingested bacteria remain confined within membrane vesicles called phago-
somes, which get swiftly flooded with acid as well as aggressive chemicals and enzymes that kill and
degrade the bacteria. The same occurs in our phagocytes, which are an essential part of our immune
system (see slide 9.3.7).
8 1 Introduction
maintain stable blood nutrient concentrations, which is important for the well-
being of the more sensitive and fastidious cells in the other organs.
2. The bacteria that reside in the large intestine produce ammonia and other
toxic metabolites, which are cleared by the liver. In patients with liver failure,
these toxic metabolites spill over into the systemic circulation, which among other
things will lead to disturbances of cerebral function. The detoxifying activity of
the liver also affects many drugs; the inactivation of drugs by the liver immediately
following intestinal uptake is known as the first pass effect (see slide 18.1.3).
The large vein that drains all the blood from the intestines and channels it
to the liver is the portal vein; together with its tributaries, it forms the portal
circulation. Aside from the intestines, the pancreas and the spleen also have their
blood drained into the portal vein.
In addition to the blood carried by the portal vein, which is at least par-
tially oxygen-depleted, the liver also receives a direct supply of oxygen-rich blood
through the liver artery. The two feeds branch out in parallel throughout the liver
and eventually merge within the tissue of the liver lobules (see below), from which
all blood is then drained toward the venous side of the general circulation.
1.5 What is the portal circulation, and which organs participate in it?
A B
C
Portal vein branch
Sinusoid
Notes: The liver has a peculiar tissue structure that is optimized for rapid and
efficient solute exchange between the percolating blood and the liver cells. While
in the tissues of most organs the blood is contained in capillaries with clearly
defined boundaries and walls, the liver has a sponge-like structure that permits
direct contact of the blood plasma with the liver cells.
A: The liver is organized into functional units called lobules, which measure
~2 mm across. In this tissue cross section, several lobules are demarcated by
strands of connective tissue that are stained red.
1.6 The digestive system 9
B: Blood from branches of the portal vein and of the liver artery percolates
each lobule and flows towards its central vein, which drains it into the general
circulation. Bile duct branches drain bile from each lobule toward the intestine.
C: Higher magnification shows the sponge-like structure of the liver tissue. In
life, blood flows through the sinusoids, which in this tissue section are visible as
the voids between strands of liver cells. The intimate contact of the liver tissue
with the percolating blood maximizes the rate of solute exchange between cells
and blood plasma.
1.6.4 Blood flow and bile flow within the liver lobule
Notes: The epithelial cells in each liver lobule are arranged in parallel layers. The
basolateral side of each cell faces the blood-filled sinusoid, while the apical side
faces a bile duct tributary. These finest, uppermost bile duct branches are so thin
that they can only be visualized using special histological techniques or by electron
microscopy.
The liver cells extract solutes from the blood, modify them, and export them
either back into the bloodstream or directly into the bile. This process is very
efficient; with some solutes, extraction and modification is almost complete during
a single pass through the liver.
1.6 Liver tissue is organized into functional units. What is the name of such a unit, and
what does its function entail?
• HCl, pH 1–2
• secreted by specialized cells in the mucous membrane (parietal cells)
• kills germs contained in food; patients with lack of gastric acid are at increased
risk of intestinal infection
• denatures food proteins and makes them accessible to cleavage by proteases
Notes: The activity of the HCl-secreting parietal cells is controlled by histamine
H2 receptors; accordingly, H2 receptor blockers such as ranitidine are effective in
10 1 Introduction
the suppression of acid secretion. Another class of drugs used to the same end
inhibit the ATP-dependent proton pump that actually brings about the secretion
of acid.
Once upon a time, excessive secretion of gastric acid was considered the main
cause of gastric and duodenal ulcers. We now know that that the true cause of
ulcers is the bacterium Helicobacter pylori, and accordingly we treat this disease
with antibiotics. Nevertheless, inhibitors of gastric acid secretion continue to be
used as well, since gastric acid aggravates the ulcers and disturbs their healing.
Individuals that lack gastric acid, due either to a disease or to drugs that
inhibit acid secretion, are more susceptible to orally contracted infectious diseases
such as cholera, Salmonella enterocolitis, and intestinal tuberculosis.
Notes: At very low pH, a protein molecule will become extensively protonated
and thereby accumulate positive charges. The mutual repulsion of these positive
charges will destabilize the protein and cause it to unfold. In this unfolded form,
all the peptide bonds become exposed and accessible to proteases.
Protein digestion is initiated right away in the stomach by the protease pepsin,
which is produced by the stomach mucous membrane. The peptide fragments will
no longer refold, even after the pH has reverted to slightly above neutral values in
the small intestine. Peptide digestion can therefore continue and be completed by
the pancreatic proteases and peptidases encountered there.
While most proteins will be unfolded by gastric acid, there are exceptions; an
obvious and important one is pepsin itself. Similarly, the coat proteins of many
pathogenic viruses, for example poliovirus or hepatitis A virus, are fairly resistant
to gastric acid as well. These viruses are therefore able to traverse the stomach
intact and then infect the mucous membranes of the intestine.
1.7 What is the physiological function of gastric acid?
1.6 The digestive system 11
6
If you already have some lab experience, you may have treated cells with trypsin or fragmented
DNA with pancreatic DNAse, and may remember that these enzyme work best at pH 7.5–8.
7
An exocrine gland secretes outwardly; this definition includes secretions into the digestive tract.
An endocrine gland secretes into the bloodstream. The products of endocrine glands are invariably
hormones.
8
Bile acids solubilize fat (triacylglycerol) effectively because they are detergents with a high
critical micellar concentration (see slide 10.2.2). For the same reason, they are also useful for
removing tough stains from your laundry.
12 1 Introduction
The greater share of the bile acids is taken up again in the lowermost section
of the small intestine, that is, the terminal ileum. Via the portal vein, they return
to the liver, where they are extracted and again secreted.
1.8 Describe and distinguish the roles of pancreatic juice and of bile in the digestion of
foodstuffs.
Notes: The small intestine comprises, from top to bottom, the duodenum, the
jejunum, and the ileum. Small substrate molecules produced by the digestive
enzymes within the gut are taken up by active transport across the mucous mem-
brane of the small intestine. The capacity for substrate uptake is obviously related
to the surface area. Accordingly, the mucous membrane is highly folded so as
to maximize the surface available for substrate uptake. This slide illustrates how
surface maximization is realized all hierarchical levels of tissue and cell structure.
The inner surface of the small intestine has circular folds, which in turn are
covered by villi. The individual epithelial cells that cover the villi are, on their
luminal surfaces, covered by microvilli. The blood that perfuses the villi of the
intestinal mucosa (red arrow) and carries away the absorbed nutrients is drained
toward the liver via the portal vein (see slide 1.6.2).
Notes: These microscopic pictures of the mucous membrane illustrate the villi
and microvilli in the small intestine. The left panel shows a low-power view of
a section across a circular fold, which is covered by a dense mane of villi. The
right panel shows an electron-microscopic image of microvilli atop an individual
epithelial cell.
1.6 The digestive system 13
...O O O
OH OH OH
. . .O O O O O O O. . .
OH OH OH OH OH OH
Notes: In the small intestine, amylose and amylopectin are broken down by pan-
creatic amylase. The main product is maltose, which is produced from amylose
and from the linear α(1 4) stretches of amylopectin. Isomaltose originates from
the α(1 6) branching points of amylopectin.
CH2 OH
O
OH
CH2 OH CH2 OH HO
O O OH
OH OH
HO O OH CH2 O
OH OH O
OH
HO OH
OH
Maltose Isomaltose
14 1 Introduction
The two disaccharides are cleaved to glucose by maltase and isomaltase, respec-
tively. These enzymes are anchored to the surfaces of the epithelial cells of the
intestinal mucosa. The same epithelial cells then take up glucose by active trans-
port (see next slide).
2 Na+ 2 Na+
SGLT1 GLUT2
Notes: After digestion, the metabolites have to be taken up by the epithelial cells
at the inner surface of the small intestine. In most cases, nutrients are taken
up by active transport, which can transport solutes energetically uphill, that is,
against their concentration gradients. Active transport is necessary to ensure the
quantitative uptake of the nutrients.
In the case of glucose, active transport is driven by the simultaneous uptake
of two sodium ions per molecule of glucose. This coupling is effected by the
SGLT1 transporter. Sodium (secreted as bicarbonate) is plentiful in the gut lumen,
while its concentration is low inside the cells. An additional driving force is the
membrane potential: the cytosol is electrically negative relative to the extracellular
space. The uphill transport of glucose is therefore driven by the simultaneous
downhill movement of sodium. Similar transporters exist for other sugars, e.g.
galactose, and for amino acids and nucleosides.
On the basolateral side of the intestinal epithelia—that is, the side that faces
the surrounding tissue, not the gut lumen—glucose is released into the extracel-
lular space, from where it can freely diffuse into the bloodstream to reach the
liver. The export from the epithelial cells is mediated by GLUT transporters.
These operate by passive transport, also known as facilitated diffusion (see slide
3.5.1). In other organs, GLUT transporters mediate the uptake of glucose. GLUT
transporters are found in all cells of the body (see section 3.5).
1.9 Explain how starch is processed in the small intestine.
1.10 Which forces affect the transport of glucose by the SGLT?
1.7 Answers to practice questions 15
• Anaerobic milieu—99% of all bacteria in the large intestine are strict anaerobes
• Bacteria degrade non-utilized foodstuffs, reducing osmotic activity of gut con-
tent
• Mucous membrane recovers water and electrolytes
• Bacterial metabolism releases potentially toxic products (e.g. ammonia), which
are taken up and inactivated by the liver
Notes: The cumulative volume of the fluids secreted into the stomach and the
small intestine exceeds four liters per day. It falls to the large intestine to recover
most of that fluid. This inevitably slows down the transport of the gut contents,
which in turn will cause them to be overgrown with bacteria.9 The bacterial flora
is mostly harmless, though, and it even helps with breaking down undigested
remnants in the gut content and thereby freeing up the water bound osmotically
by them. They produce some vitamins, too, for example folic acid, but also some
potentially toxic substances such as amines and ammonia. The latter are taken up
and dealt with by the liver.
9
The party trick that prevents bacterial colonization of our other hollow organs is to discharge
and replace the fluids more rapidly than the bacteria can grow. Accumulation and stasis of fluid
invariably leads to bacterial overgrowth and often infection; examples are recurrent urinary tract
infections when bladder function is impaired, and the respiratory infections facilitated by viscous,
slowly flowing bronchial secretions in patients with cystic fibrosis.
16 1 Introduction
1.8: Pancreatic juice supplies the depolymerizing enzymes for all foodstuffs, whereas bile
supplies no enzymes but contains bile acids, which solubilize fat but are not important
in the processing of other foodstuffs. Both pancreatic juice and bile contain bicarbonate,
which serves to neutralize gastric acid.
1.9: Starch is broken down by pancreatic amylase to maltose and isomaltose, both of
which are cleaved to glucose by cognate disaccharidases located at the surface of intestinal
epithelial cells. The epithelia take up glucose by sodium cotransport and release it at the
basolateral side through facilitated diffusion.
1.10: SGLT mediates the uptake of glucose into cells by sodium co-transport. The trans-
port is driven by three forces: (1) The concentration gradient of glucose itself—glucose can
be higher or lower outside the cell than inside, favouring or disfavouring uptake (2) The
concentration gradient for sodium; [Na+ ] is always higher outside the cell, which favors
co-transport (3) The membrane potential, which is always negative inside in non-excitable
cells such as those containing SGLT transporters; this also favors uptake.
Chapter 2
Refresher
This chapter reviews some key concepts from second year biochemistry. Feel free
to skip it if you remember a thing or two from that distant past.
As with all proteins, the activity of enzymes depends on the precise arrangement
and interaction of their amino acid residues and side chains. A straightforward
example of this is chymotrypsin. Chymotrypsin is one of the major proteases in
the human digestive tract, where its job is to knock down large protein molecules
into small peptides that are then further processed by peptidases.
Ser195
O
H
N
His57
N
H ⊖O O
Asp102
Notes: The active site of chymotrypsin contains aspartate 102, histidine 57, and
serine 195. The aspartate and the histidine cooperate to deprotonate the hydroxyl
17
18 2 Refresher
group of the serine, which then attacks the substrate peptide bond (see next slide).
Molecular structure rendered from 1afq.pdb.
The Asp-His-Ser motif is very common among proteases and esterases, so
much so that it is often simply referred to as the catalytic triad. For example, the
protease trypsin and several lipases that occur in human metabolism also have this
motif and share the same mechanism of catalysis. Other enzymes, for example
the proteasome, may contain glutamic acid instead of aspartic acid, or threonine
instead of serine. These variants still contain the same functional groups and work
the same way.
O R2
His57 Asp102
O⊖ O
Ser195 N N
H
O
H
R1
H
His57 Asp102 N C′
N′ C
O
O O⊖ R2
N NH
O⊖ Ser195
R1
O H2 N C′
N′ C
O R2
Ser195
Notes: After it has been deprotonated by aspartate and histidine, the serine per-
forms a nucleophilic attack on the carbonyl group of the substrate peptide bond.
This produces a short-lived tetrahedral intermediate that gives way when the C-
terminal peptide fragment leaves; the N-terminal fragment remains covalently
attached to the serine. This state of affairs, which is shown as the final stage in
this slide, is reached after the first half of the reaction.
In the second half reaction, which is not shown, the aspartate and histidine
residues deprotonate a water molecule, and the hydroxide ion thus formed then
bounces the N-terminal peptide fragment off the serine, again by nucleophilic
attack on the carbonyl group. The bond undergoing cleavage at this stage is an
ester, which yields more readily than the amide bond in the first stage.
2.1 What is the catalytic triad, and how does it work?
2.2 How enzymes work: active sites and catalytic mechanisms 19
With chymotrypsin, the enzyme molecule and its amino acid side chains supply
all the necessary tools for catalysis. In contrast, many other enzyme molecules
require coenzymes for their activity. For example, alanine aminotransferase, which
transfers the α-amino group from alanine to α-ketoglutarate, contains the coen-
zyme pyridoxal phosphate within its active site. In the reaction, the coenzyme
cooperates with a lysine reside that is part of the enzyme itself (see slide 12.2.1).
Most enzyme molecules have just one active site, or, in case they are mul-
timeric, one active site per subunit. However, there are exceptions: Fatty acid
synthase has as many as six different active sites on each subunit (see slide 10.5.2).
Pyruvate dehydrogenase is a multienzyme complex that contains one active site
on each subunit, but it combines three different types of subunits, each with a
different coenzyme and catalytic function, into one functional assembly (see slide
5.2.2).
Notes: The IUBMB nomenclature divides all enzymes into six classes according to
the reactions they catalyze. Within each of these main classes, there are subclasses
and sub-sub classes, which reflect differences in substrate usage and mechanism
of catalysis. The categories at all three hierarchical levels are assigned unique
numbers, and each individual enzyme receives a number as well that is unique
within its sub-sub class. An enzyme can therefore be unequivocally identified
by a dot-separated identifier containing four numbers overall. This identifier is
prefixed with the letters “EC” (for “Enzyme Commission”). Fittingly, the identifier
EC 1.1.1.1 goes to the single most important enzyme in student lifestyle—namely,
alcohol dehydrogenase, or, as IUBMB puts it, alcohol:NAD oxidoreductase.1 A list
1
This commendable enzyme, residing in the liver, degrades ethanol, and without it, some of us
might be drunk all the time!
20 2 Refresher
With each enzymatic reaction, as with any other chemical reaction, energy comes in
with these two questions: 1) will the reaction proceed at all in the desired direction,
and 2) if it does, will it proceed at a sufficient rate?
The first question is decided by the free energy of the reaction, ∆G; a reaction
will go forward if, and only if, the associated ∆G is negative. The second question
depends on the activation energy, ∆G∗ , which forms a barrier between the initial
state and the final state of the reactants. The very short-lived, energy-rich state
at the top of this barrier is called the transition state. Enzymes can substantially
lower the activation energy ∆G∗ and thus accelerate reactions, but they cannot
change the overall free energy ∆G—and therefore, the direction or equilibrium—of
the reaction.
Notes: The different roles of ∆G and ∆G∗ in biochemical reactions can be illus-
trated with a simile. The slide shows two natural lakes in the German Alps. The
Walchensee is situated 200 m above the Kochelsee. A conduit was dug across the
barrier between these two lakes to make the water flow downhill and drive a hy-
droelectric turbine. Additional tunnels drain other lakes and rivers to enhance the
supply of water to the Walchensee.
2.3 Energetics of enzyme-catalyzed reactions 21
altitude energy
difference in altitude between energy difference between
lakes metabolites (∆G)
height of ridge between lakes ∆G∗ of uncatalyzed reaction
tunnels enzymes
tunnel barrages regulatory switches of
enzymes
Notes: Our simile illustrates some, but not all aspects of enzyme reactions. For
example, all tunnels are alike; in contrast, each enzyme needs a specific “trick” or
catalytic mechanism in order to accomplish the specific task at hand. Investigating
the catalytic mechanisms of individual enzymes is an important and fascinating
aspect of biochemistry.
Another difference concerns the energetic states. The energy difference be-
tween the two lakes is completely determined by their difference in altitude. How-
ever, the difference in free energy (∆G) between two metabolites also depends
on entropy, which is determined by their concentrations. Reactions are therefore
subject to equilibrium; states with higher ∆H are less populated, but never totally
unoccupied. The equilibrium is given by this relationship:
22 2 Refresher
n1 ∆G
= e− RT (2.1)
n2
where n1 and n2 represent the numbers of molecules in the high and low energy
states, respectively. (R is the gas constant, whereas T is the absolute temperature.)
Equation 2.1 applies to the occupancy of the initial and the final states of a
reaction. It also applies to the distribution of molecules between the initial state
and the transition state of a reaction. The tendency of molecules to spontaneously
populate states of higher energy explains that chemical reactions will occur at all,
even though the energy level of the transition state is always higher than those
of the initial and final states. However, the higher the activation energy, the more
rarefied the transition state will become. The number of molecules that can first
climb the barrier and then hop down on the other side thus becomes smaller, and
the reaction slower with increasing activation energy. Enzymes—and catalysts
in general—create transition states that are lower in energy and therefore more
populated than the uncatalyzed ones.
2.3 Explain the different meanings and implications of ∆G and ∆G∗ for the equilibrium
and rate of a chemical reaction, and the effect of an enzyme on each.
As we have seen, enzymes alone cannot drive endergonic reactions forward; how-
ever, an enzyme may couple an intrinsically endergonic reaction to an exergonic
one, so as to make the overall reaction exergonic also. Most commonly, the auxil-
iary exergonic reaction consists in the hydrolysis of ATP to ADP or AMP. While this
use of ATP pervades all of enzymology, it is important to understand that there
is no equally general chemical mechanism of ATP utilization: each enzyme needs
to find its own way of actually, chemically linking ATP hydrolysis to the reaction
which it needs to drive.
Notes: As an example, this slide shows how glutamine synthetase uses ATP to
produce glutamine from glutamate and ammonia.
While the net turnover of ATP is hydrolysis, the ATP molecule isn’t hydrolyzed
directly. Instead, the phosphate group is first transferred to the substrate to create
an intermediate product, glutamyl-5-phosphate. In this mixed anhydride, the
phosphate group makes a very good leaving group, which facilitates its subsequent
substitution by ammonia. Therefore, the utilization of ATP is a central part of this
enzyme’s catalytic mechanism. We will see some more examples of ATP usage in
enzyme catalysis in the remainder of this notes.
2.4 How does ATP utilization facilitate the glutamine synthetase reaction?
2.5 Regulation of enzyme activity 23
O NH2 O⊖ O− O− O−
−O P P P Adenosine
C C C C C O O O
H H2 H2
−O
O O O O
Glutamate
ADP
C C C C C O− C C C C C
H H2 H2 H H2 H2
−O
O Pi −O
O
NH3
Glutamyl-5-phosphate Glutamine
Just as a hydroelectric power station has to adjust to variations in water supply and
demand for electricity, metabolic pathways and enzymes must adapt to changes in
substrate availability and in demand for their products. The activities of enzymes
are regulated at different levels. Activating gene expression will increase the
abundance of an enzyme, whereas activation of protein breakdown will decrease it.
In addition, there are mechanisms for reversibly activating or inactivating existing
enzyme molecules, which enable swifter and potentially less wasteful adaptation.
These reversible mechanisms are discussed in the following slides.
O− O− O−
O P O− O P O− O P O−
O O CH2 OH ATP O O O
HO HO
OH OH
OH ADP OH
Fructose-6-phosphate Fructose-1,6-bisphosphate
2.5.2 The adenylate kinase reaction equilibrates AMP, ADP and ATP
Notes: When ATP is consumed and ADP levels rise, some ATP can be regenerated
by adenylate kinase, which turns two ADP molecules into one ATP and one AMP.
According to the law of mass action, this also means that AMP levels rise quadrat-
ically with the level of ADP (assuming that changes to the level of ATP are small,
which is usually the case). Its steeper rise makes AMP a better sensor of cellular
energy demand than ADP itself, and it therefore makes sense that AMP, not ADP
regulates the activity of phosphofructokinase.
2.5 Explain how the cellular levels of AMP, ADP and ATP are related to each other.
the body of the protein to the active site and increase the efficiency of catalysis
there.
Allosteric regulation of enzymes is exceedingly common; it is not limited to
nucleotides or any other particular class of metabolites. Allosteric effectors can
be either stimulatory, as is AMP in this example, or inhibitory. As an example
of the latter, ATP is not only a cosubstrate but also an allosteric inhibitor of
phosphofructokinase.
Considering that the main purpose of the degradative pathway downstream
of phosphofructokinase is regeneration of ATP, it makes sense to reduce the
substrate flow through this pathway when ATP levels are high. The allosteric effect
of ATP on phosphofructokinase is an example of feedback inhibition, that is, the
inhibition of an early step in a pathway by that pathway’s main product. This is a
very common principle in metabolic regulation.
Notes: An allosterically regulated enzyme has two possible conformations that are
in equilibrium with each other. Both the active site and the allosteric binding site
change shape along with the molecule. An allosteric activator will bind selectively
to the regulatory site in the shape that it assumes in the enzyme’s active confor-
mation; the binding energy will shift the equilibrium towards this conformation.
Conversely, an inhibitor will selectively bind and stabilize the enzyme’s inactive
conformation.
As you can see from these considerations, activators and inhibitors may share
the same regulatory site; with phosphofructokinase, this applies to ATP and AMP.
Note, however, that human phosphofructokinase has an additional allosteric site
that permits regulation by another effector (see slide 7.5.3).
26 2 Refresher
Notes: While allosteric control is in principle feasible with both monomeric and
oligomeric enzyme molecules, almost all allosteric enzymes are indeed oligomeric
proteins. Phosphofructokinase is a dimer; this is not uncommon, but often the
2.5 Regulation of enzyme activity 27
100
Reaction velocity
75
50
25
0
0 25 50 75 100
Ligand concentration
(a) (b)
A A A A
E E
B B B B
C C C C
Notes: Cooperativity is one device for increasing the sensitivity of metabolic flux
to regulation; substrate cycles are another. This is illustrated here for activation,
but it applies similarly to inhibitory effectors as well. Let’s consider a simple,
hypothetical pathway A B C (panel a). The rate is limited by the first reaction
(A B), so that the basal throughput of both reactions is the same; we may assume
that this flow rate equals 1. If we apply an effector E that doubles the rate of the
forward reaction A B, the subsequent reaction B C will be accelerated by the
same factor, since we assumed the first reaction to be rate-limiting.
28 2 Refresher
We can achieve the same basal throughput for B C as in (a) using a substrate
cycle between A and B, in which one enzyme converts A to B with a flow rate of 2,
and a second enzyme converts B back to A with a rate of 1 (panel b). If we now add
the same effector as in (a) and accordingly double the flow rate A B, we obtain a
flow rate of 4 for A B. Diminished by the unchanged flow B A, the resulting net
flow rate for B C becomes 3. The substrate cycle therefore amplifies the increase
in metabolic flux in response to the same regulatory effect of E. This regulatory
mechanism could be made even more effective by subjecting the step B C to
inhibition by E; such a pattern is observed for example with the substrate cycle
formed by phosphofructokinase and fructose-1,6-bisphosphatase (slide 7.5.3).
Substrate cycles occur in several places in metabolism; we will see some exam-
ples in sections 6.10 and 7.5. In order to crank such a cycle, some energy must
be expended—for example, the forward reaction may hydrolyze ATP, while the
reverse reaction does not regenerate it. This energy expenditure becomes the net
effect of the cycle, and for this reason substrate cycles are also referred to as futile
cycles. The energy is simply dissipated as heat, which to a degree may be useful,
particularly in warm-blooded animals; however, the throughput of such cycles
must always be kept in check in order to avoid excessive energy wastage.
• transcriptional induction
• accelerated mRNA degradation
• ubiquitin ligation, followed by proteolytic degradation
Notes: While all of the mechanisms discussed above reversibly modulate the ac-
tivity of existing enzyme molecules, an enzyme’s activity may also be varied by
changing its abundance in the cell. Firstly, the transcription of the gene encod-
ing the enzyme can be turned on or off. This mechanism is employed by many
hormones, for example thyroid hormones or cortisol and other steroid hormones.
Similarly, the stability of the messenger RNA encoding the enzyme can be up-
or downregulated by RNA-binding proteins and other mechanisms [1], with the
corresponding effects on the abundance of the enzyme molecules.
Enzyme molecules can also be tagged with a small protein named ubiquitin,
which marks the protein for proteolytic degradation within the proteasomes.
Hormones may affect the activity of an enzyme through more than one of these
mechanisms. For example, insulin increases the activity of glycogen synthase
by way of transcriptional induction, increasing mRNA stability, and inhibition of
protein phosphorylation.
In the following chapters, we will discuss the details of regulation only with
some selected enzymes. Nevertheless, please keep in mind that virtually all en-
zyme molecules are subject to one or more regulatory mechanisms. The impor-
tance of these molecular control mechanisms in the regulation of metabolism as a
whole cannot be overstated.
2.6 Answers to practice questions 29
Glycolysis
Pathway Function
31
32 3 Glycolysis
although some is found in other tissues also. Glycogen is synthesized when glu-
cose supply is high, and its degradation helps to maintain the blood glucose level
when we are fasting. When glycogen is depleted, more glucose is synthesized from
scratch in gluconeogenesis. This pathway’s most important substrates are amino
acids, which are obtained either from a protein-rich diet—for example, when we
feast on meat exclusively—or, during starvation, from breakdown of cellular pro-
tein, mainly in skeletal muscle. Gluconeogenesis occurs in the liver and in the
kidneys.
glycolysis
glucose pyruvate
triacylglycerol
pyruvate
dehydrogenase CO2
“H2 ”
O2 ADP + Pi
respiratory chain
ATP
H2 O
mitochondrion
Notes: As noted above, glycolysis is only the first stage of glucose degradation.
Under aerobic conditions, most of the pyruvate formed in glycolysis undergoes
complete oxidative degradation to CO2 and H2 O.
Pyruvate destined for complete degradation is transported to the mitochon-
dria, where it is decarboxylated to acetyl-CoA by pyruvate dehydrogenase. Acetyl-
CoA is completely degraded in the citric acid cycle (or tricarboxylic acid cycle;
TCA cycle for short). The “H2 ” that is produced here is not gaseous but bound to
cosubstrates, as NADH + H+ and FADH2 , respectively. It is subsequently oxidized
in the respiratory chain; it is in this final stage of glucose breakdown that most of
the ATP is actually produced.
If glucose is available in excess of immediate needs and glycogen is already
stocked up to capacity, it will still be broken down by glycolysis and pyruvate
dehydrogenase to acetyl-CoA. However, acetyl-CoA will then not be oxidized, but
it will instead be used for fatty acid synthesis; the fatty acids are converted to
triacylglycerol. Fatty acid synthesis occurs in the cytosol of cells in the liver and
fat tissue.
We will now consider all these pathways in their turn, starting in this chapter
with glycolysis.
3.2 Reactions in glycolysis 33
3.1 Name the pathways involved in glucose utilization and summarize their functions.
HO OH HO HO
OH OH OH
Notes: Glucose occurs in α and β ring forms that are anomeric at the C1 carbon
(highlighted). In the presence of water, one form can reversibly change into the
other via an open-chain aldehyde form. Polymers of glucose contain either the
α or the β ring form. Within most of these polymers, the C1 hydroxyl groups
participate in glycosidic bonds, which prevents ring opening, and therefore the
spontaneous transitions between the anomeric forms cannot occur. Starch and
glycogen consist of α-d-glucose and yield it during degradation, and this form is
also the first substrate in glycolysis.
O−
4
O P O−
3-Phosphoglycerate
O
ATP ADP NADH+H+ NAD+
COO− O O OH
5
OH O OH O OH O O O
7 6
O P O− O P O− Pi O P O− O P O−
O− O− O− O−
1,3-Bisphosphoglycerate Glyceraldehyde-3- P Dihydroxyacetone- P
O P O− O P O− O OH
9 10 11
OH O− CH2 O− CH3 CH3
Metabolic reactions are catalyzed by enzymes. Enzymes are not magicians but
sophisticated catalysts, and their chemical mechanisms are often understood quite
well, at least in principle. We will now look at the catalytic mechanisms of several
enzymes from glycolysis. Some of these mechanisms will recur in similar form in
enzymes from other pathways.
2
Aldolase is sometimes referred to as aldolase A, in order to distinguish it from aldolase B, which
occurs in fructose degradation (see slide 4.2.1)
3.3 Mechanisms of enzyme catalysis in glycolysis 35
Our first example is hexokinase, which carries out the first reaction in the
glycolytic pathway. The kind of reaction that it carries out—the transfer of the
terminal phosphate group from ATP onto a hydroxyl group on the substrate—is
very common in biochemistry, and we will see more examples in these notes. The
mechanism of phosphorylation is always the same, so it suffices to discuss it once.
3.3.1 The phosphate groups in ATP are shielded from nucleophilic attack
NH2
R X⊖ N
N
⊖O ⊖O ⊖O
⊖ ⊕
O P O P O P N N
O
O O O
OH OH
Notes: Most reactions that involve the transfer of a phosphate group from ATP to
something else are exergonic, that is, they are energetically favorable. For example,
the hydrolysis of ATP—that is, the transfer of the phosphate group to water—has
a free energy of -35 J/K mol. It is interesting to note then that ATP is nevertheless
quite stable in solution, which means that there must be a high activation energy
barrier that resists hydrolysis of the phosphate anhydride bond.
This energy barrier is due to the negative charges at the perimeter of the
triphosphate group that shield the phosphorus atoms in the center from nucle-
ophiles which are also negatively charged. Accordingly, to lower this barrier,
kinases provide compensating positive charges within their active sites that en-
gage the negative charges on the ATP molecule and thereby clear the way for
nucleophilic attack on the phosphorus.
Notes: In hexokinase, the negative charges of the phosphate groups are shielded
by magnesium ions and by positively charged amino acid side chains in the active
site. This facilitates nucleophilic attack by and phosphate group transfer to the
substrate.
The charge shielding mechanism solves one problem, but another one remains,
namely, how to limit the reaction to the right nucleophile. After all, the most
abundant nucleophile in the cell is water, which should make hydrolysis the most
likely outcome of ATP activation. This problem is addressed in the next slide.
36 3 Glycolysis
H
NH N
Arg
O O O
R X⊖
O O O
R X P O− −O P O P Adenosine
O− O− O−
Notes: After binding of its substrate glucose (yellow and red) and its cosubstrate
ATP (not shown), hexokinase adopts a closed conformation, in which substrate
and cosubstrate are buried within the enzyme, and water is excluded from the
active site and from the reaction (left). The magnified view on the right shows the
C6 oxygen of glucose peeking out of the enzyme’s active site; ATP would occupy
the cavity above. Rendered from 3b8a.pdb.
H2 C OH H OH
O O
Glucose OH OH Xylose
HO OH HO OH
OH OH
If the enzyme is given xylose instead of glucose, one water molecule can squeeze
into the active site along with the sugar. This water assumes the place of the C6
hydroxymethyl group of glucose, and it will be activated by hexokinase to react
with ATP, which will result in ATP hydrolysis.
3.3 Explain the catalytic mechanism of hexokinase.
3.3 Mechanisms of enzyme catalysis in glycolysis 37
His His
⊕
N N Lys
P H P ⊕NH3 P
Lys
O OH OH H⊕
OH H ⊕NH3 OH H O H OH OH O
HO O H ⊖OH HO HO H ⊖O O
OH OH OH
Glu
P P P
O OH OH
OH OH H OH
C OH OH
HO CH2 OH HO HO
H H O O
OH O O
H
Glu
His
Cys Cys
NADH
S O O H⊕ S O
O
H O P O−
R ⊖O P O−
R O−
O−
genation reactions, for example in the citric acid cycle and in the β-oxidation of
fatty acids. The reaction goes through the following steps:
1. A cysteine in the enzyme’s active site is deprotonated by an auxiliary histidine
to a thiolate anion (−S– ).
2. Glyceraldehyde-3-phosphate (shown as R−(− −O)−H) binds to the active site,
and the thiolate performs a nucleophilic attack on its aldehyde carbon. This
yields a tetrahedral intermediate state, in which the substrate becomes bound
covalently to the enzyme—hence the term “covalent catalysis”.
3. The covalent intermediate gives up two electrons and two protons to NAD+
and the enzyme, which yields NADH and converts the substrate to a thioester.
NADH leaves.
4. The thioester is cleaved by a phosphate ion, again through nucleophilic attack.
The product (1,3-bisphosphoglycerate) leaves, and the enzyme is restored to
its original state.
The redox cosubstrate used by glyceraldehyde-3-phosphate dehydrogenase,
nicotinamide adenine dinucleotide (NAD+ ), also accepts most of the hydrogen that
accrues in the degradative pathways downstream of glycolysis. Its structure and
the details of its reduction by hydrogen are shown in slide 3.3.6.
3.4 Summarize the catalytic mechanisms of phosphohexose isomerase and of glyceralde-
hyde-3-phosphate dehydrogenase.
O− R
Notes: The panel on the left shows the structure of NAD+ and NADP+ ; the re-
dox-active nicotinamide moiety is highlighted. The panel on the right shows the
reduction of this moiety by glyceraldehyde-3-dehydrogenase. The electrons and
the hydrogen are transferred from the substrate to the C4 of the nicotinamide. The
electrons then redistribute within the ring.
3.3 Mechanisms of enzyme catalysis in glycolysis 39
As you can see, NAD+ and NADP+ differ solely by the absence or presence of
a phosphate group at the lower ribose ring, which has absolutely nothing to do
with the actual redox chemistry. Why, then, is it there at all? It simply serves as
a tag that allows NAD+ and NADP+ to interact with separate sets of enzymes. The
significance of this duality is discussed in a later chapter (see slide 9.3.1).
N
N
O O− O−
−O P O− ⊖O P O P O N N
O
O O O
⊕H
O
CH2 OH OH
O−
H
O O
O O
CH2 CH3
O− ⊕H O−
Notes: As you know, the most abundant and important energy-rich metabolite
in the cell is ATP. Within this molecule, the energy is stored in the energy-rich
phosphate anhydride bonds. Cleavage of these bonds is exergonic, and the energy
released by cleavage drives the various reactions and processes powered by ATP.
Conversely, in creating ATP from ADP, we require energy to form a new phosphate
anhydride bond. We just saw how this energy is derived from phosphoenolpyru-
vate, and we thus may say that the enolphosphate group is another energy-rich
group.
The first ATP in glycolysis is formed by cleavage of the carboxyphosphate
mixed anhydride in 1,3-bisphosphoglycerate, and we may thus infer that such
mixed anhydrides are energy-rich groups, too. The same functional group also
occurs in acetylphosphate and in succinylphosphate, and both of these are
capable as well to drive the formation of phosphate anhydride bonds in ATP or
GTP.
In slide 3.3.5, we saw that the carboxyphosphate was formed from ionic phos-
phate and a thioester bond. Since the free phosphate ion is low in energy, it follows
that the energy that went into the mixed anhydride came from the thioester. This
means that thioesters are energy-rich groups, too.
NAD+ NADH + H+
Carrier-H2 Carrier
1/2 O2 H2 O Mitochondrion
NAD+ NADH + H+
COO− COO−
H OH O
CH3 CH3
Lactate Pyruvate
Notes: Anaerobic glycolysis also occurs in many microbes, which also face the
need to reoxidize NADH. Without the option of reverting to oxidative metabolism
within a short time span, they must also deal with the continued accumulation of
acid. The yeast Saccharomyces cerevisiae solves this problem through ethanolic
fermentation: The acid is converted to a neutral and considerably less toxic com-
pound (ethanol) via decarboxylation. The CO2 developed in this reaction makes
3
Measurement of the blood lactate concentration is performed in sports medicine to gauge the
capacity of a trained athlete to sustain aerobic rather than anaerobic metabolism during prolonged
exertion. The anatomical correlate of endurance is not so much the quantity of muscle tissue but
the extent of its vascularization, that is, the abundance of capillaries in the tissue. A high density of
capillaries ensures good oxygen supply.
42 3 Glycolysis
bread dough rise up, whereas the ethanol does the same to government tax rev-
enue.4
NAD+ NADH + H+
C C COO−
H2 C OH HC O O
CH3 CH3 CH3
3.6 Explain how the NADH that accumulates in the glyceraldehyde-3-dehydrogenase reac-
tion is oxidized under anaerobic conditions in humans and in baker’s yeast.
3.5 Transport and utilization of glucose in the liver and in other organs
As was noted in slide 1.6.13, uptake of glucose from the blood occurs by facilitated
diffusion and is mediated by GLUT transporters. These occur in several sub-types
whose properties are tuned to the physiological roles of different organs.
Notes: A typical carrier for passive substrate transport alternates between two
conformations that are open to either side of the membrane. On both sides of
the membrane, the substrate reversibly binds to the carrier protein; binding and
dissociation are governed by mass action kinetics. Transport occurs when the
protein changes from the outward-facing to the inward-facing conformation, or
vice versa, while a substrate molecule is bound to it.
The sequence of events in passive transport resembles the pattern of Michaelis-
Menten enzyme kinetics—in both cases, a solute binds reversibly to a protein,
which then performs some state-altering action on it. Accordingly, the velocity of
4
The English name of this organism is baker’s yeast, but the literal translation would be “sugar
fungus of the beer”.
3.5 Transport and utilization of glucose in the liver and in other organs 43
[S]
Vtransport = Vmax (3.1)
KM + [S]
3.5.2 GLUT transporters in different tissues vary in their affinity for glucose
100
Transport rate (% of max)
GLUT3 (brain)
75
GLUT4 (muscle, fat)
50
GLUT2 (liver)
25
0
0 2 4 6 8 10
Glucose (mM)
performs the same reaction as does hexokinase but differs from the latter by a
higher KM value.5
100
50
25 Glucokinase (liver)
0
0 2 4 6 8 10
Glucose (mM)
Accordingly, in the liver, phosphorylation will proceed more slowly when the
level of blood glucose is low, and most of the glucose will be allowed to pass
through the liver and make its way into the general circulation. In contrast, at
high concentration, the liver will extract a greater share of the available glucose
and convert it to glycogen or fatty acids. The kinetic properties of both glucose
transport and phosphorylation therefore support the regulatory function of the
liver in blood glucose metabolism.
At this point, we must note that the intrinsic kinetic properties of transporter
and enzyme molecules are but one piece in the puzzle of glucose regulation. In-
sulin and several other hormones control the expression, distribution and activity
of transporters and enzymes. Hormonal regulation is essential for proper coordi-
nation of glucose utilization, as is evident from its severe disturbances in diabetes
mellitus and other endocrine diseases (see chapters 13 and 14).
The graphs in this slide and the previous one were plotted using parameters
tabulated in references [4, 5, 6].
3.7 Explain how glucose uptake and utilization is prioritized between different tissues.
Notes: Starch is the most abundant carbohydrate in our diet, which makes glucose
the most important dietary monosaccharide. However, our diet contains several
other sugars in significant amounts. The guiding motif in the metabolism of these
sugars is economy: instead of completely separate degradative pathways, there
are short adapter pathways which merge into the main pathway of carbohydrate
degradation, that is, glycolysis.
Lactose and sucrose are disaccharides. Degradation of both sugars begins with
hydrolytic cleavage, which releases glucose and galactose or glucose and fructose,
respectively. Fructose is also found in the diet as a monosaccharide. We already
know how glucose is degraded, so we here only need to concern ourselves with
the remaining monosaccharides. The degradation of sorbitol will be discussed as
well, whereas ribose and deoxyribose will be covered in later chapters.
47
48 4 Catabolism of sugars other than glucose
Notes: Sucrose is produced from sugar cane and sugar beet, which contain it in
high concentrations (15–20%). In a typical Western diet, it may amount to as
much as 20% of the total carbohydrate intake. Sucrose consists of glucose and
fructose joined by a β-glycosidic bond between the carbon 1 of glucose and carbon
2 of fructose.
The hydrolytic cleavage of sucrose, like that of of maltose, occurs at the surface
of the intestinal epithelial cells. The enzyme responsible is β-fructosidase, also
named sucrase. Both sugars are then taken up by specific transport: Glucose
by the SGLT1 transporter, and fructose by the GLUT5 transporter, which is named
after glucose but actually transports fructose more effectively than glucose.
OH 1 OH 2 OH
OH OH
Fructose Fructose-1- P
3
O O
4
OH OH O
OH ATP ADP O P O−
O−
Glyceraldehyde Glyceraldehyde-3- P
Notes: Fructose degradation, also called fructolysis, runs mostly in the liver. In
the first step, fructose is phosphorylated by fructokinase (1), which uses ATP as
a cosubstrate. This yields fructose-1-phosphate. The latter is then cleaved by
aldolase B (2). The products of this reaction are dihydroxyacetone phosphate,
which is already a metabolite in glycolysis, and glyceraldehyde, which can enter
glycolysis after phosphorylation by glyceraldehyde kinase (4).
4.2 Degradation of fructose and sucrose 49
Fructokinase Aldolase B
Fructose Fructose-1- P Glyceraldehyde
Dihydroxyacetone- P
ATP ADP
Glyceraldehyde-3- P
NADH+H+ NAD+
1
Haima is the Greek word for blood; hematology is the medical discipline that deals with diseases
of the blood.
50 4 Catabolism of sugars other than glucose
OH OH
Lactose Galactose
Lactase
Galactokinase
Glycolysis
...
would accomplish the same net effect, without being chemically more difficult in
any way.2
4.3 Summarize the Leloir pathway of galactose degradation.
UDP-Galactose OH
O
CH2 OH
O NH
O OH
O O
O P O P O N O
O
OH O− O−
OH
O
CH2 OH
O NH
OH
O O
HO O P O P O N O
O
OH O− O−
UDP-Glucose OH
Notes: A deficiency of the lactase enzyme in the small intestine gives rise to
lactose intolerance, which is found frequently in people of East Asian descent who
are past their infant age.
If lactose is not cleaved, it cannot be absorbed, so it travels down the drain
from the small to the large intestine. Many of the bacteria found there have the
capacity to metabolize lactose, which they will happily convert to acids and gas.
For example, Escherichia coli has a pathway called mixed acid fermentation. One
2
Galactose is contained in the glycosyl moieties of many glycoproteins and glycolipids. The
enzymes and activated intermediates for the synthesis of galactose from glucose and for its incor-
poration into glycosyl moieties are widespread among life forms. They predate the emergence of
lactose secretion by mammals, and evolution chose to reuse them for lactose utilization.
52 4 Catabolism of sugars other than glucose
of the products of this fermentation is formic acid (HCOOH), which is then cleaved
by formic acid lyase to H2 and CO2 . The decarboxylation of formic acid serves the
same purpose as that of pyruvate in ethanolic fermentation, namely, the removal
of excess acidity resulting from the fermentation (see slide 3.4.3).
The aberrant fermentation and gas formation leads to abdominal discomfort and
diarrhea. Since the environment in the large intestine lacks oxygen, H2 generated
in the bacterial fermentation is not oxidized but instead enters the system as such
and is mostly exhaled. An increase in exhaled hydrogen gas provoked by ingesting
a test dose of lactose can be used to diagnose the condition.
Treatment consists in omission of lactose in the diet. Milk can be pre-treated
with purified bacterial β-galactosidase, rendering it suitable for consumption by
lactose-intolerant individuals. Fermented milk products such as yogurt and cheese
are depleted of lactose by microbial fermentation and therefore do not pose a
problem for lactose-intolerant individuals.
4.4 Explain the pathogenesis of lactose intolerance.
4.3.4 Galactosemia
Notes: Three different enzyme deficiencies in the pathway are subsumed under
the name galactosemia, which means “galactose in the blood.” All of these are
rare; type I is the most common and most severe form. Here, the deficient enzyme
is galactose-1-phosphate uridyltransferase. This leads to a buildup of galactose-
phosphate, but also of several other metabolites. The disease becomes manifest
in newborns with acute liver failure and is deadly if not promptly diagnosed and
treated. In many countries, this enzyme defect is part of neonatal screening
programs.
4.4 Sorbitol is an intermediate of the polyol pathway 53
I galactose-1-phosphate- galactose,
uridyltransferase galactose-1-phosphate,
galactitol, galactonate
II galactokinase galactose, galactitol
III UDP-galactose galactose-1 phosphate,
epimerase UDP-galactose
Therapy consists in the removal of galactose from the diet, but even so organ
damage develops, most commonly affecting the CNS and, in girls, the ovaries. The
residual pathology that develops in spite of the diet is ascribed to the endogenous
synthesis of galactose, which proceeds via UDP-glucose and UDP-galactose; the
UDP-galactose epimerase reaction is reversible.
For a long time, it was assumed that accumulation of galactose-1-phosphate
and phosphate depletion are responsible for cell and organ damage, which is
analogous to the pathogenic mechanism in fructose intolerance (see slide 4.2.2).
However, this assumption has been thrown into question by the results of animal
experiments. When galactose-1-uridyltransferase is genetically knocked out in
mice, these develop a profile of metabolite accumulation that closely resembles
human patients, but they do not display any of the pathology observed in humans
[8]. What is more, some rare human cases have been reported that show the usual
biochemical manifestations, but no clinical signs [9]. The quest for the true cause
of the pathology affecting most human patients continues [10, 11].
In the order of the pathway, type II galactosemia comes first, as it involves a
defect of galactokinase. In this case, galactose simply does not enter the Leloir
pathway at all; it builds up in the blood and is mostly eliminated in the urine.
The liver will not be adversely affected. However, there is a common complication
elsewhere: the eyes will develop cataract, that is, obfuscation of the lenses. This
is due to the reduction of galactose to galactitol in the cells of these organs by
aldose reductase (see slide 4.4).
The rarest form of galactosemia is due to the defect of UDP-galactose epimerase.
The biochemical pattern is similar to type I, except that UDP-galactose also ac-
cumulates, and as in type I, developmental delay seems to occur [12]. In this
condition, both the utilization and the synthesis of galactose are inhibited, and
it appears necessary to maintain a low level of dietary galactose to supply the
synthesis of galactose-containing glycolipids and glycoproteins.
4.5 Describe and discuss type I galactosemia.
Notes: Sorbitol is not strictly a sugar, since it has no keto or aldehyde group. It
is normally a minor component of dietary carbohydrates, but it is also prepared
semisynthetically and used as a sweetener. In addition, it is formed in our own
54 4 Catabolism of sugars other than glucose
metabolism from glucose in the polyol pathway, which then converts it further
to fructose. Note the use of NADPH as a cosubstrate in the first step, and of
NAD+ in the second. NADP exists mostly in the reduced state, while NAD is mostly
oxidized (see slide 9.3.1), which means that both drive the pathway in the indicated
direction.
O OH OH
NADPH+H+ NADH+H+
OH OH O
HO HO HO
OH OH OH
NADP+ NAD+
OH OH OH
OH aldose OH sorbitol OH
reductase dehydrogenase
glucose sorbitol fructose
The first enzyme, aldose reductase, is not specific for glucose but can also reduce
galactose, which gives rise to galactitol. As stated above, the elevated blood level
of galactose in galactosemia type II causes galactitol to accumulate in the lenses;
the same occurs with glucose and sorbitol in insufficiently treated diabetes mellitus
(see slide 14.5.7). Accumulation of either sorbitol or galactitol causes cataract; this
is ascribed to their osmotic activity, which causes cell damage through swelling.
Like the cells in the lens, nerve cells are able to take up glucose in an insulin-in-
dependent fashion, and like cataract, nerve cell damage (diabetic polyneuropathy)
is a common long-term complication in diabetes. It appears plausible that sorbitol
accumulation might also be responsible for this nerve cell damage. Inhibitors of
aldose reductase have been developed and have shown promise in animal models
of both diabetes and galactosemia, but evidence of clinical effectiveness in humans
is scarce.
Conversion of glucose to fructose via the polyol pathway occurs in the seminal
vesicles, which are part of the male sexual organs, and fructose is found in the
sperm fluid. It supplies the sperm cells with fuel in their frantic quest for an
oocyte; the advantage of this somewhat unusual source of energy may be that
fructose will not be pilfered by the other tissues the sperm fluid will get into
contact with.
Now, if sperm cells require fructose to sustain their motility, one might expect
that prevention of fructose synthesis with aldose reductase inhibitors would dis-
rupt male fertility and thus provide the long-sought pill for males. However, I have
not seen any experimental studies on this subject.
tosemia results from a defect of fructokinase and causes a buildup of free fructose in
blood and urine.
4.3: Galactose undergoes phosphorylation to the 1-phosphate by galactokinase. Galactose-
1-phosphate is exchanged for glucose-1-phosphate by galactose-1-phosphate uridyltrans-
ferase, which uses UDP-glucose as its other substrate. Glucose-1-phosphate is converted to
the 6-phosphate by phosphoglucomutase, whereas UDP-galactose epimerase regenerates
UDP-glucose.
4.4: Lactose intolerance results from a deficiency of intestinal lactase. Ingested lactose
cannot be cleaved and taken up in the small intestine and therefore reaches the large
intestine, where it is metabolized by bacteria. Changes in bacterial metabolism, including
increased gas formation, gives rise to abdominal discomfort and diarrhea.
4.5: Type I galactosemia is caused by a defect of galactose-1-phosphate uridyltransferase.
Metabolites upstream of the deficient enzyme accumulate, as do the aberrant conversion
products galactitol and galactonate. The disease causes potentially severe pathology in the
liver and other organs, but the biochemical mechanism of organ damage is not precisely
understood.
Chapter 5
5.1 Overview
H+
Pyruvate Mitochondria
CO2
Acetyl-CoA
Oxaloacetate Citrate
Malate Isocitrate
CO2
H2
Fumarate α-Ketoglutarate
CO2
Succinate Succinyl-CoA
O2 ADP + Pi
ATP
H2 O
57
58 5 Pyruvate dehydrogenase and the citric acid cycle
Notes: Pyruvate is produced by glycolysis in the cytosol, while PDH and all sub-
sequent degradative steps are located in the mitochondria. Therefore, pyruvate
needs to be transported from the cytosol to the mitochondrial matrix.
The outer mitochondrial membrane contains porins, which are membrane
proteins that form non-specific pores and allow free permeation of most small
metabolites, including pyruvate. In contrast, the inner mitochondrial membrane
is much more restrictive, and it is permeable to only those metabolites for which
it contains specific carrier systems. The pyruvate carrier is an active transporter
that co-transports pyruvate and a proton.1
Red blood cells and blood platelets lack mitochondria and accordingly cannot
degrade pyruvate. These cells reduce pyruvate to lactate, which they then release
into the bloodstream (see slide 3.4.2).
5.1 Summarize the metabolite transport properties of the inner and outer mitochondrial
membranes.
5.2.1 The PDH reaction occurs in three successive steps that are catalyzed by
three different subunits
O OH
H3 C O CoA–SH NAD+
E1 E2 E3
H3 C O
This reaction does not occur all at once; instead, it is carried out as a sequence
of group transfers and redox steps by three different catalytic subunits. These
subunits are named according to the specific partial reactions they catalyze:
1. pyruvate dehydrogenase2 removes CO2 ,
1
Alternatively, we might say that pyruvate is exchanged for an OH– ion; the net effect is virtually
the same.
2
You will notice that the name “pyruvate dehydrogenase” is ambiguous, denoting both the entire
complex and the first subunit.
5.2 Structure and function of pyruvate dehydrogenase 59
E1 E1 + E2 E1 + E 2 + E 3
5.2.3 A lipoamide tether guides the substrate from one active site to the next
Notes: The catalytic efficiency of PDH is further increased by another neat trick:
The intermediate substrates become covalently attached to lipoamide. This coen-
zyme is covalently attached to E2 , but due to its long, flexible linker can reach into
the active sites of adjacent E1 and E3 subunits as well. The lipoamide tether thus
guides the substrate from one active site to the next, preventing it from leaving
until it has completed the course.
5.2 Describe the structural organization of pyruvate dehydrogenase, and explain the role
of the covalently bound dihydrolipoyl moiety.
3
The pyruvate dehydrogenase complex remains intact when purified from cell extracts. With
some other enzymes, there is evidence that they form functional complexes in vivo, even though they
emerge from purification procedures as individual and functional molecules. For example, malate
dehydrogenase and citrate synthase may associate in vivo, so that oxaloacetate may pass directly
from one to the other [13]. Another intriguing example is the association of glycolytic enzymes with
the outer surface of the mitochondria [14].
60 5 Pyruvate dehydrogenase and the citric acid cycle
O NH
S S
H
O
E1 E3
E2
Notes: Each of the subunits E1 –E3 requires a coenzyme to work its particular magic.
In addition, two cosubstrates are also used, namely, NAD+ and coenzyme A.4
Notes: The coenzyme thiamine pyrophosphate (TPP), which is derived from thi-
amine (vitamin B1 ), is associated with the E1 subunit of PDH. In cooperation with
an aspartate residue in the active site, TPP forms a carbanion, that is, a negative
charge on a carbon atom. The TPP carbanion is resonance-stabilized; an electron
can move back and forth between the carbanion and the neighboring cationic
nitrogen.
Carbanions are very powerful nucleophiles, and the TPP carbanion functions
as such in the decarboxylation of pyruvate.
4
Yes, strictly speaking, coenzyme A is a cosubstrate, since it is transformed from one state to
another in the reaction; a true coenzyme should emerge from the reaction unchanged, just like
the enzyme. As you can see, the distinction between coenzymes and cosubstrates is not strictly
maintained in the traditional nomenclature.
5.2 Structure and function of pyruvate dehydrogenase 61
Asp Asp
O O
O O⊖
H
⊕ H ⊕
N N N N
H S P S P
N N H P N N H P
H
H
⊕
⊕ N N
N N
⊖ S P
S N NH2
N N H P
H2
Notes: The TPP5 carbanion attacks the keto group of pyruvate, which leads to a
covalent intermediate from which CO2 is cleaved. This yields another carbanion,
now located within the hydroxyethyl group that is the remainder of the substrate.
This new carbanion, which is again resonance-stabilized by TPP, sets the stage for
the next step in the reaction.
R1 R1 R1
⊕ R2 ⊕ R2 ⊕ R2
N N N
⊖ S S S
⊖O ⊖O
O
C C CH3 C C CH3 ⊖C CH3
C
O O O O
O O
H⊕ H H
Notes: The plot continues from the previous slide at the top right. The carbanion
on the hydroxyethyl group attacks the disulfide bond of lipoamide. The entire
substrate is then cleaved from TPP and carried by lipoamide to E2 , where it is
transferred to coenzyme A and released as acetyl-CoA. Lipoamide is reduced
to dihydrolipoamide in the process; it is reoxidized by E3 , which transfers the
hydrogen to NAD+ , using FAD as an intermediate carrier. This concludes the
pyruvate dehydrogenase reaction.
5.3 Give an overview of the three steps involved in the PDH reaction.
5
Note that only the thiazole ring of TPP is shown in this slide; its second ring is represented by
R1 .
62 5 Pyruvate dehydrogenase and the citric acid cycle
E3
NADH+H+
FAD E1
NAD+ R1
H
S H FADH2
S ⊕ R2
N
⊕ S
H
H3 C S ⊖C CH3
S
S O
O CoA H
E2
SH SH O
CoA S⊖ S S
⊕H
CH3
H3 C O
We had seen earlier that enzymes may be regulated by allosteric control or through
phosphorylation (section 2.5). Pyruvate dehydrogenase exemplifies both mecha-
nisms.
PDH
5.4.1 The overall reaction of the TCA cycle: does it add up?
Notes: While the substrate carbon enters the TCA cycle as acetyl-CoA, the coen-
zyme A moiety is simply hydrolyzed off in the very first reaction; therefore, with
only a little sleight of hand, we can neglect coenzyme A and substitute acetate for
acetyl-CoA as the substrate.
If we look back at figure slide 5.1.1, we see that the TCA cycle produces four
molecules of H2 and two molecules of CO2 . Now, if we attempt to balance our
single acetate substrate with these amounts of CO2 and H2 , we see that we are
short 4 hydrogen and 2 oxygen atoms on the left side. However, we can balance
the equation by adding two molecules of water.6
What this means is that half of the H2 produced in the TCA cycle is gained
by the reduction of hydrogen from water. The water-derived oxygen that accrues
in the process is used to complete the oxidation of the acetate carbon. Hydrogen
derived from both water and acetate is then re-oxidized in the respiratory chain to
generate ATP.
The energy yield of the TCA cycle itself, in terms of directly generated energy-
rich phosphoanhydride bonds, is very modest—just one molecule of GTP, equiva-
lent to one ATP, is generated for each molecule of acetyl-CoA degraded, compared
to approximately 15 in the respiratory chain. This comparison shows that the TCA
cycle’s main contribution to ATP generation is to provide H2 for the respiratory
chain.
Notes: The first reaction in the TCA cycle is catalyzed by citrate synthase. It is
mediated by acid-base catalysis; abstraction of a proton from the methyl group
of acetyl-CoA by an aspartate residue in the active site converts acetyl-CoA to an
enol form, which then attacks the carbonyl group of oxaloacetate. The reaction is
assisted by two histidine residues and pulled forward by the subsequent hydrolysis
of citryl-CoA. Figure drawn after a scheme given in [15].
6
Where exactly do the two water molecules enter the TCA cycle? For one of them, it is obvious
(see step 7 in slide 5.4.3). However, the second one is a bit harder to spot. Hint: it is not the H2 O
that hydrolyzes off the acetyl-CoA in the citrate synthase reaction, since that is already accounted
for by substituting acetate for acetyl-CoA in our simplified reaction scheme.
5.4 The citric acid cycle 65
Acetyl-CoA
His274
CoA His274
⊕ CoA
S O H N
N S O H N
N
O O⊖ H H
CH2
H
Asp375 H2
C COO−
− OOC
Oxaloacetate
O
H
CoA N
CoA-SH H2 O ⊕
HO O S O His320
N
CH2 CH2
− OOC C COO− − OOC C COO−
H2 H2
OH OH
Citrate Citryl-CoA
Notes: The paragraph numbers below correspond to those of the reactions in the
figure. The first reaction in the figure is the second in the cycle overall, which is
why it gets the number 2.
2. The hydroxyl group in the newly formed citrate is shifted to an adjacent
carbon to yield isocitrate. This reaction is catalyzed by citrate isomerase and
involves the transient elimination of water across the two carbons involved; the
water is then added back in the reverse orientation.
3. Isocitrate is decarboxylated and dehydrogenated by isocitrate dehydroge-
nase, which yields α-ketoglutarate. In contrast to the pyruvate dehydrogenase
reaction, dehydrogenation precedes decarboxylation. The dehydrogenated inter-
mediate is known as oxalosuccinate.
4. α-Ketoglutarate is converted to succinyl-CoA by α-ketoglutarate dehydro-
genase. This catalytic mechanism of this enzyme is completely analogous to that
of pyruvate dehydrogenase.
5. Succinyl-CoA is converted to succinate by succinate thiokinase, and GDP is
concomitantly phosphorylated to GTP. From the mechanism of the glyceraldehyde-
3-phosphate dehydrogenase reaction (Figure 3.3.5), we already know that thioester
bonds are energy-rich and can drive the phosphorylation of carboxylic acids. A
carboxylic acid phosphate, succinylphosphate, also occurs as an intermediate in
the succinate thiokinase reaction. As is the case with 1,3-bisphosphoglycerate, the
phosphate group is subsequently transferred to a nucleotide diphosphate. While
succinate thiokinase uses GDP rather than ADP, the amounts of energy involved
are virtually the same.
6. Succinate is dehydrogenated across the CH2 −CH2 bond by succinate dehy-
drogenase, which yields fumarate. The coenzyme used in this reaction is flavin
adenine dinucleotide (FAD). As a rule of thumb, you can assume that FAD is used
66 5 Pyruvate dehydrogenase and the citric acid cycle
in the dehydrogenation of CH−CH bonds, whereas either NAD+ or NADP+ are used
in the dehydrogenation of CH−OH bonds. While all other enzymes in the TCA
are in aqueous solution in the mitochondrial matrix, succinate dehydrogenase is
bound to the inner surface of the inner mitochondrial membrane; it is identical
with complex II of the respiratory chain (see slide 6.4).
Citrate Isocitrate
NAD+
3
Succinyl-CoA α-Ketoglutarate NADH+H+
4
H2 C COO− NADH+H+ NAD+ H2 C COO− H+ H2 C COO−
3
CH2 CH2 HC COO−
Pi CoA
5
CoA-SH Succinate Fumarate
CH2 CH2
5 6
O O O O− − OOC H
−O P O H2 O
O− 7
− OOC
8
H − OOC H
Oxaloacetate Malate
Notes: As pointed out above, α-ketoglutarate dehydrogenase uses the same cat-
alytic mechanism as pyruvate dehydrogenase. The similarity is reflected in a high
degree of homology between the subunits of the two enzymes. If you look closely
5.5 Regulation of the citric acid cycle 67
at the PDH mechanism (Figure 5.2.1), you will see that the reactions carried out by
the final subunit (E3 ) will be identical in both cases, since E3 comes into play when
only hydrogen is left but the rest of the substrate has already been disposed of.
Indeed, the two multienzyme complexes share the very same E3 protein; only E1
and E2 are specific for the respective substrates. The same E3 subunit occurs yet
again in an analogous multienzyme complex that participates in the degradation
of the branched chain amino acids (slide 12.4.4), as well as in the glycine cleavage
system (slide 15.2.6).
Pyruvate α-Ketoglutarate
E1 E1
CO2 CO2
CoA-SH CoA-SH
E2 E2
Acetyl-CoA Succinyl-CoA
NAD+ NAD+
E3 E3
+ +
NADH+H NADH+H
Several metabolites in the citric acid cycle are also substrates in biosynthetic
pathways, for example those for heme or various amino acids, and through these
pathways are drained from the cycle. When this occurs, they will need to be
replenished. Similarly, when the workload of a cell and its ATP demand increase,
the TCA cycle must then process acetyl-CoA at an accelerated rate, which requires
an increase in the pool of TCA cycle intermediates.
The first thing to note is that just feeding more acetyl-CoA into the TCA
cycle does not address this problem, since acetyl-CoA simply offsets the two CO2
molecules that are lost in subsequent reactions in the cycle. Instead, we need a net
supply of any of the intermediates with four or more carbon atoms that function
catalytically rather than as substrates.
One important and abundant source of TCA cycle intermediates is the pyruvate
carboxylase reaction, which makes oxaloacetate from pyruvate (slide 7.2.4). Often,
however, the oxaloacetate thus obtained is immediately diverted again toward
glucose synthesis (gluconeogenesis). In this situation, amino acids become the
major net source of TCA cycle intermediates (see chapter 12).
In the active site of E2 , the acetyl group is transferred from lipoamide to coenzyme A.
Lipoamide becomes reduced to dihydrolipoamide in the process; it is reoxidized by E3 ,
which transfers the H2 to NAD+ .
5.4: PDH is subject to allosteric activation by fructose-1,6-bisphosphate and to inhibition
by acetyl-CoA and NADH. It is also subject to inhibition by phosphorylation, which in turn
is activated by the products and inhibited by the substrates of PDH.
5.5: Yes. Metabolism without structures is as useful as memorizing phone books (or
probably even less so).
5.6: NAD-dependent isocitrate dehydrogenase is inhibited by ATP and NADH. The latter
also inhibits α-ketoglutarate dehydrogenase and shifts the malate dehydrogenase equilib-
rium away from oxaloacetate, which slows down the citrate synthase reaction.
Chapter 6
6.1 Introduction
In the respiratory chain, the NADH and FADH2 that was accumulated in the pre-
ceding degradative pathways is finally disposed of by reacting it with molecular
oxygen. The free energy of this “cold combustion” is used to generate ATP. The
amount of ATP generated in the respiratory chain far exceeds the modest quan-
tities produced in the upstream pathways; this is the reason why only aerobic
metabolism enables us to sustain physical exertion for extended periods of time.
The workings of the respiratory chain are quite different from all other path-
ways in human metabolism. Each of those other pathways consists of a succession
of discrete enzymatic reactions. Inasmuch as these pathways contribute to the
production ATP, the energy is always passed from one energy-rich bond to the
next, with a newly created phosphoanhydride bond in ATP as the final recipient. In
contrast, the respiratory chain combines chemical reactions with physical forces
that are not pinned down to individual molecules, and the energy is stored and
converted in novel ways.
71
72 6 The respiratory chain
6.2 Overview
Notes: The respiratory chain involves four large protein complexes (I–IV) as well as
ATP synthase (AS). All of these are embedded in the inner mitochondrial membrane.
Coenzyme Q (Q) and cytochrome C (C) are diffusible electron carriers.
H⊕ H⊕ H⊕
C
Q ⊖ ⊖
I III IV AS
⊖ ⊖
II
ADP + Pi
In this scheme, the mitochondrial matrix is below the membrane, whereas the
cytosol is above it. The reactions carried out in the chain are explained below.
Note that the reactants in this scheme are not stoichiometrically balanced.
1
As mentioned before, the porins in the outer mitochondrial membrane are permeable for most
small molecules and ions, and thus the proton concentration in the space between the two mito-
6.3 ATP synthesis can be separated from electron transport 73
If you think that all this sounds somewhat strange and vague, you are quite
right—but don’t let that trouble you. The purpose of this overview is only to divide
and conquer, to break up the overall process into manageable parts that we can
then tackle in detail in their turn.
The first thing to note about electron transport and ATP synthesis is that they can
be experimentally separated from one another. So-called uncoupling agents allow
the observation of electron transport without ATP synthesis. On the other hand,
ATP synthesis works without electron transport if a proton gradient is created in
some other way.
H⊕ H⊕ H⊕
C
Q ⊖ ⊖
⊖ ⊖
H⊕
Notes: Uncoupling proteins are membrane proteins, also embedded in the inner
mitochondrial membrane, that passively transport protons, allowing them to by-
pass ATP synthase. Electron transport and hydrogen oxidation—and, upstream
of it, degradation of energy-rich substrates—will continue, but ATP synthesis will
cease; the energy that would have gone into ATP synthesis is simply dissipated as
heat.
Uncoupling proteins are found in particularly high concentration in the mi-
tochondria of brown fat tissue, which differs from the more abundant white fat
tissue by its high density of mitochondria.2 Brown fat tissue serves the purpose
of producing heat, by way of simply oxidizing fat without ATP production. The
physiological significance is discussed in slide 10.3.8.
chondrial membranes equilibrates readily with the cytosol. The proton concentration gradient that
powers ATP synthesis therefore exists across the inner mitochondrial membrane only.
2
The cytochromes of the abundant mitochondria give this tissue its brown color; the white color
of regular fat tissue tells us that its density of mitochondria must be low.
74 6 The respiratory chain
OH OH
NO2 NO2
NO2 NO2
H⊕ H⊕
O⊖ O⊖
NO2 NO2
NO2 NO2
Notes: The synthetic compound dinitrophenol can diffuse across the inner mi-
tochondrial membrane in both its protonated and unprotonated form. It can
therefore carry protons into the mitochondrion, thereby dissipating the driving
force for ATP synthase.3 Just like uncoupling proteins do in brown fat tissue, dini-
trophenol will induce the burning of fat or other substrates and the production
of heat—but in the mitochondria everywhere, not just in brown fat tissue. In the
1930s, dinitrophenol was used as a drug for losing excess body weight. It worked,
too, but cases of fatal hyperthermia and other side effects caused the speedy dis-
continuation of this use. Wikipedia reports that the drug remains popular among
bodybuilders, however, which seems of a piece with other reckless self-medication
practices among the followers of this cult.
Notes: The effects of uncouplers show that electron transport can occur in the
absence of ATP synthesis. On the other hand, ATP synthesis will occur in the
absence of electron transport if a proton gradient is sustained in some other
way. This was elegantly demonstrated by Ephraim Racker, who incorporated ATP
synthase into liposomes along with bacteriorhodopsin, a membrane protein from
the halophilic (that is, salt-loving) bacterium Halobacterium halobium.
Bacteriorhodopsin is a very unusual protein that functions as a light-driven
proton pump: just shining light on it makes it pump protons across the membrane.
In our experiment, illuminating the sample will make bacteriorhodopsin pump
3
Dinitrophenol is a hydrophobic molecule, and it therefore is not surprising that it can cross
membranes in protonated form. However, the high membrane permeability of its deprotonated,
negatively charged form is unusual. This is related to the two electron-withdrawing nitro groups,
which cause the negative charge to be highly delocalized; this prevents the molecule from attract-
ing a tightly bound hydration shell that otherwise would interfere with its permeation across the
membrane.
6.4 Molecules in the electron transport chain 75
protons into the liposome. This will crank the ATP synthase and make it synthesize
ATP from ADP and ionic phosphate.
ADP+Pi
H⊕
H⊕
H⊕ H⊕
H⊕ H⊕
H⊕
H⊕
ATP
H⊕
Note that the orientation of both proteins shown here is inside-out relative to that
found in their natural host membranes. Therefore, protons will here accumulate
on the inside, and ATP synthesis proceed on the outside; this is the reverse of the
situation in vivo.
The theoretical significance of these experimental findings is that, although
the electron transport chain and ATP synthase reside in the same membrane and
in close proximity, the proton gradient is the only required functional link between
them. The electron transport chain generates the proton gradient, whereas ATP
synthase puts it to work and thereby dissipates it. Because of this clear distinction,
we can safely examine these two functions separately from each other.
6.1 Explain the theoretical implications of dinitrophenol uncoupling of the respiratory
chain, and of the Racker experiment.
Notes: This slide shows the structures of the four respiratory chain complexes that
form the respiratory chain.4 The gray rectangle represents the inner mitochondrial
membrane. Among the redox cofactors, yellow and red blobs represent iron sulfur
clusters. Organic rings (black) with red balls (iron atoms) in the center are hemes;
other organic rings are flavins or ubiquinone (Q). The blue ball next to one of the
hemes in complex IV is the copper ion in its active site. Each of the four complexes
has a specific role in the electron transport process:
1. Complex I accepts hydrogen from NADH + H+ and is therefore also called
NADH dehydrogenase. The NADH is oxidized back to NAD+ and thereby readied
for the next round of reduction in the TCA cycle or by pyruvate dehydrogenase.
The hydrogen is split into electrons and protons. The electrons travel along a
string of redox cofactors that traverses the entire protein complex and are then
transferred to the small, membrane-resident carrier molecule known as ubiquinone
4
Drawn from 3m9s.pdb, 2fbw.pdb, 3cx5.pdb, 2zxw.pdb, and 3cyt.pdb, after a figure in [16].
76 6 The respiratory chain
Mitochondrial matrix
Cytoplasmic side
cytochrome C
the electrons to flow in the right direction, the successive transitions must be
exergonic, that is, their free energy (∆G) must be negative.
In the figure above, you can see a multitude of redox cofactors, neatly spaced
along the protein molecules, that function as “stepping stones” for the migrating
electrons. These prosthetic groups fall into various structural classes.
N⊖ N
Fe2⊕
⊖
N N
HO
Notes: Most of the redox cofactors in the respiratory chain are hemes and iron-
sulfur clusters, both of which contain iron ions. Hemes are tetrapyrrol rings that
hold a single central iron ion. This iron ion can adopt different oxidation states,
mostly Fe2+ and Fe3+, although Fe4+ occurs with one of the hemes in complex IV.7
In iron-sulfur clusters, it is again the iron that accepts and donates electrons
by alternating between different oxidation states. Each iron ion is held in place by
four sulfur atoms, which either belong to cysteine side chains (orange) or are free
sulfides (S2– ; yellow). The two types of sulfurs may occur in various proportions,
which results in iron-sulfur clusters of different size.
The pyrrole rings or sulfur atoms do not only keep the iron ions in place but
also modulate their redox potentials. These potentials are further tweaked by the
specific molecular environment of each cofactor, in such a way as to enable the
electrons to flow from one cofactor to the next.
6.2 Describe the iron-containing redox cofactors that occur in the respiratory chain.
Notes: The flavin nucleotides flavin adenine dinucleotide (FAD) and flavin mononu-
cleotide (FMN) can occur in three different states of reduction, which differ by
single electrons. Unlike NAD, which can accept or donate electrons only in pairs,
flavins can accept or yield electrons one at a time. Therefore, flavins can buffer
the electron flow between NAD and iron-containing redox cofactors, and this is
7
Some of the hemes in the respiratory chain are referred to as “cytochromes.” Somewhat con-
fusingly, the same term is, with other molecules, applied to the entire complex of a heme and the
protein it is bound to.
78 6 The respiratory chain
why the very first cofactor that accepts the electrons from NADH in complex I is
indeed an FMN molecule.8
O− R
H
O P O X H3 C N N O
X = H: FMN
OH O
X = AMP : FAD NH FMNH/FADH
OH H3 C N
OH
OH R
H
H3 C N N O H3 C N N O
NH NH FMNH2 /FADH2
H3 C N H3 C N
H
In addition to the stationary redox cofactors that occur within complexes I–IV,
there are two electron carriers that are not tightly associated with one individual
complex but function as shuttles between them:
1. Ubiquinone or coenzyme Q. This coenzyme contains a quinone group. It
carries electrons, as hydrogen, from complexes I and II to complex III. It also con-
tains a long hydrophobic polyisoprene tail, which confines it to the hydrophobic
interior of the membrane. Like flavins, ubiquinone can transfer electrons singly or
in pairs; this is important in the coenzyme Q cycle (see slide 6.6.3).
2. Cytochrome C. This is a small protein that again contains a heme. It is
located at the outer surface of the inner mitochondrial membrane and shuttles
electrons between complex III and complex IV.9
6.3 Explain the role of flavin cofactors in electron transfer processes.
Notes: Several redox cofactors in the respiratory chain are prone to side reactions
with molecular oxygen, which produce superoxide (O–2 ) and other reactive oxygen
species, that is, partially reduced forms or oxygen. These have the potential to
damage cellular membranes and macromolecules and must be scavenged. This
topic is discussed further in slide 9.3.8.
8
Electron buffering between NADPH and heme by flavins occurs in cytochrome P450 reductase
(see slide 18.2) and in nitric oxide synthase (slide 9.3.5).
9
In addition to its role in the respiratory chain, cytochrome C is also an important intracellular
signaling molecule; its release from damaged mitochondria triggers apoptosis (programmed cell
death; see reference [17] and slide 18.5.1). Another molecule with surprising connections to
apoptosis is glyceraldehyde-3-phosphate dehydrogenase [18]; this enzyme is reportedly associated
with the outer mitochondrial membrane.
6.5 The energetics of electron transport 79
R R FAD/FMN
H O2 O–•
2
N N O N N O
NH NH
N N
H+
O− O coenzyme Q
O2 O–•
2
O O
O ... O ...
O O
V V
e⊖ e⊖ 2e⊖ 2e⊖
1
2 H2 H⊕ Q Q⊖ H2 2H⊕ H⊕+NAD⊕ NADH
Notes: This slide illustrates the experimental setup for measuring the redox po-
tential of an electron carrier. Left panel: coenzyme Q withdraws electrons from
the standard hydrogen electrode, which by definition gives it a positive redox po-
tential (∆E). Right: NADH pushes electrons toward the standard electrode, making
its ∆E negative.
In the experimental setup, the molecule of interest and a reference solute
are contained in two adjacent buffer-filled chambers. Platinum electrodes are im-
mersed in both solutions and connected through a voltmeter (V). As electrons are
withdrawn from the solute in one chamber and delivered to the other, the volt-
meter indicates the direction and magnitude of the potential difference. Protons
and other ions can flow across a salt bridge between the chambers so as to pre-
serve electroneutrality. In order to allow the flow of ions but prevent mixing of the
80 6 The respiratory chain
6.5.2 The redox potential (∆E) is proportional to the free energy (∆G)
energy
∆G ≡
moles (number of molecules)
energy
∆E ≡
charge transferred
charge transferred
∆G = ∆E ×
moles
therefore
∆G = −∆E × n × F (6.1)
Notes: From the previous slide, it is clear that electrons will flow spontaneously
from one redox cofactor to another if the corresponding ∆E is positive. We also
know that reactions proceed spontaneously if their ∆G is negative. The two param-
eters are directly related to one another according to equation 6.1. Either one is
therefore sufficient to describe the energetics of the reaction; the reason why redox
potentials are more commonly used in this context is that they can be measured
more directly than ∆G.
In the equation, ∆E is the difference in the redox potentials between two co-
factors. The parameter n is the number of electrons transferred in the reaction;
for example, NADH feeds two electrons at a time into the chain, which means that
n equals two for this reaction. In contrast, heme typically accepts and donates
single electrons, which means that n = 1. The F in the equation is Faraday’s
constant, which tells us how many units of charge are carried by one mole elec-
6.5 The energetics of electron transport 81
trons (96,500 coulombs/mol).10 One can think of a cofactor’s redox potential as its
affinity for electrons—the higher it is, the more strongly the cofactor will attract
electrons.11
−0.4 75
NADH FMN
I
(Fe-S)N-2 FADH2
III
Heme c1 CytC
′
Heme a3
0.4 −75
IV
O2 −150
0.8
Notes: This slide shows the redox potentials, and the corresponding free energy
levels, of some selected electron carriers in the respiratory chain. The lowest
potential is found with NAD+ , in keeping with its position at the start of the
transport chain. The next carrier in sequence, FMN, is part of complex I. It has a
slightly higher potential than NADH and is therefore able to accept its electrons.
The redox potential increases continuously along the respiratory chain to reach its
highest value at oxygen, which therefore has the highest affinity for the electrons
and gets to keep them. Reduced oxygen, which recombines with protons to yield
water, thus is the end product of respiration.
The iron-sulfur cluster N2, which occupies the lowermost position within com-
plex I as shown in slide 6.4, has a significantly higher potential than the FMN. This
step in potential corresponds to a significant amount of free energy that is released
at some point within complex I as the electrons travel through it from FMN toward
N2. Complex I uses this energy to expel protons from the mitochondrion, against
10
Remember that the volt, which is the unit of ∆E, is defined as joule/coulomb, since voltage =
energy/charge,
hence the need for Faraday’s constant.
Like Avogadro’s number, Faraday’s constant is a relic of history, required only because the physical
units of mass and electrical charge had already been arbitrarily chosen before the inherent masses
and charges of atoms and electrons were discovered. One could in principle define a system of units
without either of these crummy numbers. Indeed, chemists often use Daltons, and physicists use
electron volts (eV), in order to avoid them.
The minus sign in equation 6.1 results from the fact that the electron-donating electrode, the
cathode, is considered negative. This is entirely arbitrary and meaningless, but it is also very handy
as a trap in exam questions.
11
You have encountered the same concept with chemical elements as their electronegativity: An
element with a high electronegativity holds on to electrons particularly tightly, i.e. it has a high
affinity for electrons.
82 6 The respiratory chain
their concentration gradient. Major steps in potential that drive proton expulsion
also occur within complex III and complex IV.
Only minor steps of potential occur in the delivery of electrons from complex I
to complex III via coenzyme Q, and between complexes III and IV via cytochrome C.
Likewise, with complex II, the potentials of both entry and exit points must fall into
the narrow interval between FADH2 and coenzyme Q, which means that very little
energy is released as electrons traverse this complex. Such minor steps in redox
potential suffice to jog the electrons along, but they are too small to contribute to
proton pumping.
6.4 What is the relationship between free energy and redox potential?
6.5 At which of the four complexes in the electron transport chain does the greatest step
in redox potential occur? Which one has the smallest step?
In addition to their specific redox potentials, which establish the general direction
of electron flow, the redox cofactors in the respiratory chain also differ in two
other important aspects:
1. NADH, FADH2 , FMNH2 and coenzyme Q carry both electrons and protons—
that is, hydrogen. In contrast, the hemes and the iron-sulfur clusters carry
only electrons.
2. NAD+ can only accept and donate pairs of electrons, whereas the hemes and
iron-sulfur clusters can only accept and donate single electrons.
The switch from the two-electron carrier NADH to the one-electron carrying Fe−S
clusters within complex I is negotiated by FMN, which, as discussed above, can
accept or donate electrons both pairwise and singly.
Notes: After accepting H2 from NADH + H+ (equation 6.2), FMNH2 donates the
electrons one by one to the first Fe−S cluster (equations 6.3 and 6.4), adopting a
sufficiently stable radical form between these two transfers. The electron transfer
between FMNH2 and Fe−S also illustrates what happens if an electron-only carrier
is reduced by a hydrogen carrier: The protons are simply shed into the solution.
6.6 Explain how electrons are transferred from NADH to the iron-sulfur clusters in com-
plex I.
6.6 Interfacing different types of electron carriers 83
O
O
e⊖
2H⊕ 2e⊖
O⊖ OH
2H⊕
O O
O ... e⊖ O ...
O OH
Q⊖ QH2
Notes: Hydrogen carriers may also be reduced by electron carriers. This happens
with coenzyme Q, which is reduced by the iron sulfur cluster N2 in complex I. In
this case, protons are taken up from the solution, but only in the second reduction
step:
Notes: The Q cycle or ubiquinone cycle runs within complex III, which accepts
reduced ubiquinone from the surrounding membrane. Complex III has two binding
sites for ubiquinone, and both of them are occupied while the cycle runs; we will
here call them A and B. The ubiquinone cycle goes through the following stages,
starting at the top left:
1. At the outset, a reduced ubiquinone (QH2 ) is bound to site A, and an oxidized
one (Q) is bound to site B. The protons of QH2 are stripped off and expelled to
the cytosolic side. The electrons part company and migrate to two different
84 6 The respiratory chain
electron carrier groups within complex III; one of these is an iron-sulfur cluster,
whereas the other is a heme.
to complex IV
2 H⊕
A A
QH2 Q – cytochrome C
QH2
Q Q e⊖
B B
to complex IV
2 H⊕
A A
Q – QH2
QH2 Q⊖
B B
2H⊕
2. The iron sulfur cluster donates its electron to cytochrome C, while the heme
transfers its electron to the second molecule of ubiquinone in site B. The
ubiquinone molecule in site A, now oxidized, trades places with another one
in the surrounding membrane that was reduced in the preceding steps of the
respiratory chain.
3. The protons and electrons of the new QH2 in site A are abstracted and split
as in the first step. The heme passes on its electron to coenzyme Q in site
B. Now completely reduced, the coenzyme Q picks up two protons from the
mitochondrial matrix to form QH2 .
4. The electron that had been transferred to the Fe–S cluster is donated to cyto-
chrome C. The Q in site A trades places with QH2 in site B, which completes
the cycle.
According to this scheme, with each molecule of ubiquinone reduced in the
respiratory chain, the two protons it carries are expelled into the cytosol, and two
additional protons are taken up from the mitochondrial matrix and expelled into
the cytosol as well. Therefore, complex III uses ubiquinone as a prosthetic group
to facilitate the movement of protons across the membrane.
If you compare the outline of the ubiquinone cycle given here to the description
in your textbook, you might find the similarity somewhat remote. In reality, as you
can see in slide 6.4, complex III contains several more redox co-factors that act as
intermediate stepping stones in the electron transfer steps outlined above. They
have been skipped here for simplicity.
6.7 How can electrons be transferred from electron-only carriers such as iron-sulfur clus-
ters to hydrogen carriers such as ubiquinone?
6.7 How is electron transport linked to proton pumping? 85
2+ 3+ 4+
e⊖
O O− O2−
O O− OH−
H⊕
1+ 2+ 2+
e⊖ H⊕
O2
2+ 3+ 3+
2e⊖ 2H⊕
OH−
OH−
H2 O
1+ 2+ 2+
• Some redox steps in the ETC are coupled to proton binding and dissociation,
which may occur at opposite sides of the membrane. Example: Coenzyme Q
cycle at complex III
86 6 The respiratory chain
• Redox steps that do not involve hydrogen directly need a different mechanism
in order to contribute to proton pumping. Example: Sequence of iron-sulfur
clusters and hemes in complex IV
Notes: As pointed out above, some of the protons that undergo expulsion from
the mitochondrion are accepted from the hydrogen carriers NADH and ubiquinone
and travel together with electrons for a part of the journey. However, at some point
they must part company, and the protons must be expelled, whereas the electrons
are retained. Also, more protons are being expelled than can be accounted for by
the hydrogen carriers. This is obvious with complex IV, which does not interact
with any hydrogen carriers at all yet expels up to four protons for each pair of
electrons accepted. So, there must be mechanisms that extract energy from the
transfer of proton-less electrons and apply it towards the expulsion of electron-less
protons. How does this work?
Notes: The experimental evidence on this point is still fragmentary, and inasmuch
as they are understood, the emerging mechanisms are quite complex. Therefore,
instead of trying to describe them faithfully, this slide presents a simplified con-
ceptual model to provide an idea of how things might work.
The basic idea is that capture and release of electrons cause conformational
changes to a protein. This is entirely analogous to conformational changes caused
by allosteric effectors binding to enzymes, or by phosphate groups bound to
cytoskeletal proteins such as the myosin light chain. An electron carries a charge,
a charge causes a field, and a field creates forces that act on charged residues on
the protein; thus, in principle, it is not hard to imagine how migrating electrons
can cause conformational changes.
6.8 ATP synthesis 87
It is commonly stated that approximately ten protons are ejected for each pair of
electrons abstracted from NADH, such that four protons are ejected at each of
complexes I and IV, and 2 at complex III.12 Complex II does not eject any protons
but just abstracts them from FADH2 and passes them on to ubiquinone. If you
look at slide 6.5.3, you will notice that the difference in the redox potentials of
FAD and ubiquinone is rather small. Consequently, the amount of free energy
associated with the transfer of electrons from FAD to ubiquinone is too small to
permit the performance of work against the proton gradient.
Most of the ATP that results from complete oxidative degradation of glucose is syn-
thesized only after the substrate has already vanished in the form of CO2 and H2 O.
At this stage, the entire available energy is stored in the so-called proton-motive
force across the inner mitochondrial membrane. ATP synthesis is powered by the
protons that yield to this force and are pulled back by it into the mitochondrion.
Notes: The proton concentration in the cytosol is approximately ten times higher
than that in the mitochondrial matrix. While this creates a significant significant
driving force, the larger contribution to the overall proton-motive force comes
from the electrostatic membrane potential across the inner mitochondrial mem-
brane. Like the proton concentration gradient, this electrical potential is a direct
consequence of the proton pumping: Each proton ejected leaves one negative
charge behind inside the mitochondrion.
In a fully energized mitochondrion, the membrane potential amounts to ap-
proximately ~150 mV.13 According to equation 6.8, this potential confers a free
energy of approximately 15 kJ/mol to each proton, whereas equation 6.7 indicates a
12
Note that this number is at variance with the mechanism of the coenzyme Q cycle given above,
which ejects four protons for each equivalent of ubiquinone. Different sources offer varying num-
bers.
13
Above a potential of 150 mV, the inner membrane becomes increasingly leaky for protons (see
section 6.10), so that such higher potentials will not be sustained.
88 6 The respiratory chain
H⊕ H⊕ H⊕ H⊕ H⊕ H⊕ H⊕
H⊕ H⊕ H⊕
X⊖ X⊖ X⊖ X⊖ X⊖ X⊖ X⊖ X⊖ X⊖ X⊖
[H+ ]in kJ
∆Gconcentration = RT ln =6 (6.7)
[H ]out
+ mol
kJ
∆Gpotential = ∆ψ × n × F = 15 (6.8)
mol
6.9 What are the two physical forces that together form the “proton-motive force” at the
inner mitochondrial membrane, and which is the dominant one?
6.10 Compare the proton-motive force of ~20 kJ/mol to the free energies of electron trans-
port at each respiratory chain complex in slide 6.5.2. What conclusions can you draw for
the plausible stoichiometry of proton ejection at each complex?
Rotation of γ
and F0 subunits
δ
α β
b1 γ
F0
c c
proton flow
Notes: This complex and fascinating molecule is both an enzyme and a molecular
motor.14 The slide shows a side view. Ten identical c subunits are arranged like
pie slices; the whole cylindrical pie is referred to as the F0 subunit. It and the a
subunit are embedded in the inner mitochondrial membrane, whereas the other
subunits of the molecule protrude into the mitochondrial matrix.
14
A similar molecular motor drives the rotation of the flagella found in many bacteria, which
enables them to swim. Remember that mitochondria are of bacterial origin.
6.8 ATP synthesis 89
The F0 and the γ subunit (both shaded in red) rotate relative to all other
subunits. The γ stalk therefore rotates within and rubs against the bushing formed
by the six α and β subunits. The rotation is driven by the flow of protons that
occurs at the interface of the a and F0 subunits.
β
γ
ATP
ADP Pi
ATP
Notes: The interaction of the rotating γ subunit with the static β subunits is
crucial to ATP synthesis. Because of the asymmetric shape of the γ subunit, its
rotation imposes cyclic conformational changes upon the α and β subunits that
surround it. The β subunit, which contains the active site of the enzyme, extracts
mechanical work from these conformational changes and ultimately converts it
into the chemical energy contained in ATP.
How does the β subunit convert mechanical into chemical energy? Strictly
speaking, it doesn’t—it turns out that the isolated β-subunit can create ATP from
ADP and ionic phosphate all by itself. In doing so, the β subunit adopts two distinct
conformational states. In the first state, it binds ADP and phosphate. Once both
are bound, β transitions to the second state, which binds ATP with exceptionally
high affinity but no longer binds ADP and phosphate. Formation of several avid
but non-covalent bonds between β and ATP provides the energy that is needed to
create the new, energy-rich phosphate anhydride bond in ATP.
With the isolated β subunit, we have at this point reached a dead end—ATP is
bound so avidly as to never be released, which means that no further turnover can
occur. It is at this stage that, in the intact enzyme, the γ subunit comes into play.
Rotation of γ forces another change of conformation upon β that in turn kicks the
ATP out of the active site. The force imposed by γ on β must be so strong as to
overcome and offset the large binding energy that ties the ATP to β. In summary,
therefore, the synthesis of ATP proceeds spontaneously inside β, and the energy
90 6 The respiratory chain
of rotation is applied to force out the avidly bound ATP and prime β for the next
round of catalysis.
6.11 Explain the binding-change model of ATP synthesis inside ATP synthase.
Notes: The last remaining puzzle is how the proton flux actually causes the mov-
ing parts of the enzyme to rotate. As shown in slide 6.8.2, the proton flux passes
between the a subunit and the F0 subunit, which contains 10 identical c chains
arranged in a pie-slice fashion; for simplicity, only one c chain is highlighted in
the current slide.
The two gray cylinders in this cartoon are proton conduits contained within
the a subunit; the remainder of the a subunit is not shown. The proton conduits
span the outer and the inner membrane leaflet, respectively, but they do not meet;
it is the rotating F0 disk that mediates the flow of protons between them. To this
end, each c chain has a strategic aspartate residue that faces the surrounding lipid
membrane and can reversibly accept a proton.
In the top left of the figure, the aspartate (shown as a little groove) of the
highlighted c subunit is about to accept a proton from the periplasmic space15
via the corresponding conduit. In the next frame, the disk has moved a bit and
accepted the proton; it then goes around by almost a full turn, until it reaches the
other conduit (bottom right). In the final frame, the proton has left the disk and
is on its way to the mitochondrial matrix, while the c subunit is about to move on
and pick up then next proton.
There is indeed evidence that agrees with this model; for example, the strategic
aspartate on the F0 disk is alternatingly accessible from the two opposite sides
of the membrane. However, while it seems simple and elegant, this model has
15
The periplasmic space is the the narrow space between the inner and the outer mitochondrial
membrane.
6.8 ATP synthesis 91
one fundamental shortcoming: While it tells us how rotation of ATP synthase can
dissipate the proton gradient, it does not explain how ATP synthase manages to
derive any torque from this process, so that it can perform work against resistance.
More specifically, what makes the rotor keep going in the same direction instead
of just oscillating back and forth between the two conduits? This would save it
the trouble of performing work, while still permitting proton flux.16
Notes: The absence of a flywheel effect that would help F0 move past dead spots
means that proton flux and F0 rotation must be tightly coupled. This assumption
is supported by the observation that the c chain undergoes a significant confor-
mational change upon protonation or deprotonation [19]. The authors of the cited
study proposed a functional model that is shown here in a simplified cartoon
depiction.
One of the 10 c subunits is highlighted in blue, as is the proton carried by it.
In the top left, this subunit is about to accept a proton from the cytosolic conduit.
In the second frame, the proton has been accepted, and the c subunit has rotated
about its own axis. The entire F0 disk then rotates by nearly a full turn, which
brings our highlighted c subunit to the inward-connecting conduit. The aspartate
now delivers its proton, whereupon the subunit swivels back about its own axis.
During this transition, the aspartate holds on to the end of the conduit—that is,
the stationary a subunit—and thus causes the entire F0 disk to rotate forward.
This second rotation of the c subunit constitutes the “power stroke” of the engine.
The model implies that the number of protons transported per rotation is
identical to that of the c subunits in F0 . Intriguingly, ATP synthases in different
16
A steam engine gets around a similar problem by employing inertia, which is provided by a nice,
heavy, cast-iron flywheel. That is not possible here because of the minuscule dimensions. A student
who took my class in 2005, Kelvin Cheung, took on the challenge to calculate the kinetic energy of
rotating ATP synthase; it works out to about one billionth of the energy required for making 1 ATP.
92 6 The respiratory chain
organisms vary in their number of c subunits, which will directly affect the sto-
ichiometric ratio of ATP synthesis to proton transport. It would be interesting
to know whether there are complementary variations in the number of protons
driven out per electron during electron transport. Otherwise, the different subunit
stoichiometry of F0 should directly translate into a different ATP yield in the entire
respiratory chain.
In slide 3.4.1, it was mentioned that, under aerobic conditions, the NAD+ con-
verted to NADH in the cytosol by glyceraldehyde-3-phosphate dehydrogenase is
re-oxidized in the respiratory chain. NADH itself, however, cannot pass the in-
ner mitochondrial membrane, and in fact not even the more permissive outer
membrane. How, then, is its oxidation accomplished?
It turns out that NADH is not translocated at all but is reoxidized, or dehy-
drogenated, in the cytosol. The hydrogen is then taken to the mitochondrion by
other carriers. These shuttle system work in a somewhat roundabout manner,
tying together several enzyme activities with specific transporters in the inner
mitochondrial membrane.
malate malate
+
NAD NAD+
NADH+H+ NADH+H+
oxaloacetate oxaloacetate
Notes: A simple but somewhat dubious shuttle is shown in this picture. Among
the various mitochondrial exchange transporters that engage malate, there is one
that can directly swap it for oxaloacetate [20]. This transporter has been found in
the mitochondria of multiple mammalian organs, and under suitable conditions
in vitro, it can sustain reoxidation of external NADH by isolated mitochondria [21].
The problem with this hypothetical shuttle is the ratio of NADH to NAD+ ,
which in vivo is usually higher in the mitochondria than in the cytosol. This should
crank the malate-oxaloacetate shuttle the wrong way, exporting NADH from the
mitochondria rather than importing it. Therefore, until evidence of its operation
within intact cells becomes available, this cycle cannot be assumed to be of major
significance.
6.9 Auxiliary shuttles for the mitochondrial reoxidation of cytosolic NADH 93
α-ketoglutarate α-ketoglutarate
2 2
aspartate aspartate
glutamate H+ H+ glutamate
Notes: The malate-aspartate shuttle combines four substrates and two trans-
porters with two enzymes, both of which are required on both sides of the mem-
brane. In the cytosol, malate dehydrogenase (1) regenerates NAD+ by reducing ox-
aloacetate to malate, which is then exchanged for mitochondrial α-ketoglutarate.
Inside the mitochondrion, malate is converted back to oxaloacetate, which is then
transaminated by aspartate aminotransferase (2). This yields aspartate, which is
exchanged for cytosolic glutamate, as well as α-ketoglutarate, which is exchanged
for malate. Transamination is then reversed in the cytosol, which restores oxaloac-
etate and glutamate and closes the cycle.
While the malate-aspartate shuttle is more complex than the malate-oxaloac-
etate shuttle, it does have the advantage with respect to driving force: the gluta-
mate-aspartate exchanger cotransports one proton with each molecule of gluta-
mate, which means that the proton-motive force drives the cycle in the required
direction [22].
The significance of this shuttle in vivo is supported by both experimental and
clinical observations. Genetic defects of enzymes or carriers in the cycle cause
lactic acidosis, that is, a pathological accumulation of lactic acid in the body,
which indicates that the normal disposal of cytosolic NADH is disrupted. Similarly,
pharmacological inhibition of aspartate aminotransferase with aminooxyacetate
inhibits glucose oxidation and increases lactate levels [22, 23].
6.12 Draw the malate-aspartate shuttle, and explain why it is more plausible to occur
in vivo than the simpler, hypothetical direct exchange of malate and oxaloacetate.
Glycerophosphate Glycerophosphate
GPD
NAD+ FAD QH2
NADH+H+ FADH2 Q
DHAP DHAP
Most of the time and in most cells, the respiratory chain runs at rates that are
substantially below the maximum. How is the flow rate of the respiratory chain
controlled? In a healthy and not maximally exerted cell, there is much more ATP
than ADP or phosphate, so that these become limiting for the flow. If ATP synthase
is short of substrates, dissipation of the proton-motive force will slow down. The
proton pumps will find it difficult to extrude more protons, and since electron
transport and proton pumping are tied to one another, the dehydrogenation of
NADH and FADH2 will slow down as well.
The flow rate of the respiratory chain also affects those of glycolysis and the
TCA cycle. Both ATP and NADH participate in this regulation:
6.10 Regulation of the respiratory chain 95
CO2 CO2
Notes: The above regulatory mechanisms are quite straightforward. There is, how-
ever, one remaining mystery. We have already noted that there are two forms
of isocitrate dehydrogenase, one using NAD+ and the other NADP+ as the cosub-
strate. While the NAD+ -dependent form is inhibited by NADH and ATP, the NADP+ -
dependent form is not, and one might thus expect that it would go at full blast
even when the demand for ATP is low and NADH is high. What is more, the NADP+ -
dependent enzyme is actually more highly expressed than the NAD+ -dependent
form. How, then, is the NADP+ -dependent enzyme prevented from uncontrolled
consumption of isocitrate, and why does it exist at all?
The answer to the first question is that, at least when demand for ATP is low,
the NADP+ -dependent isocitrate dehydrogenase reaction is close to equilibrium.
This equilibrium is sustained by a high mitochondrial level of NADPH, which in
turn is maintained by nicotinamide nucleotide transhydrogenase.
Notes: This remarkable protein, which is located in the inner mitochondrial mem-
brane, is both an enzyme and a transporter. It reduces NADP+ to NADPH at the
96 6 The respiratory chain
expense of NADH. As with ATP synthase, the enzyme reaction is coupled to the
translocation of protons:
NADH + NADP+ + H+
out NAD+ + NADPH + H+
in
NADPH + H+ NAD+
NADP+ NADH + H+
H+
Note that the transhydrogenase involves only mitochondrial NAD and NADP; the
pools existing in the cytosol are unaffected.
NADP-dependent NAD-dependent
dehydrogenase dehydrogenase
NADPH NAD+
NADP+ NADH
transhydrogenase
+
H
Notes: This slide and the following one show how the transhydrogenase may be
integrated with the function and regulation of the TCA cycle and the respiratory
chain [25].
When the demand for ATP is low, mitochondrial NADH and the proton-motive
force will both be at high levels, which will cause the transhydrogenase to reduce
NADP+ at the expense of NADH. Accordingly, the NADPH concentration will be
high, which results in near-equilibrium conditions for the NADP+ -dependent isoci-
trate dehydrogenase also. If the NADPH concentration is high enough, there would
be a low net flux within an interesting futile cycle that involves both isocitrate de-
6.11 ATP yield of complete glucose oxidation 97
hydrogenases and the transhydrogenase, and the net effect of which is the influx
of one proton in each round.17
NADP-dependent NAD-dependent
dehydrogenase dehydrogenase
NADPH NAD+
transhydrogenase
H+
Notes: On the other hand, when the demand for ATP is high, the proton-motive
force and the level of NADH will be lower. Under these conditions, the transhydro-
genase should switch direction, now consuming NADPH to produce more NADH,
which in turn will be consumed at a fast clip in the respiratory chain. This also
means that transhydrogenase will work as an auxiliary proton pump, thus aug-
menting the proton-motive force and ATP synthesis.
What is more, the consumption of NADPH by transhydrogenase will topple
the equilibrium of NADP+ -dependent isocitrate dehydrogenase. Like the NAD+ -
dependent enzyme, it will now consume isocitrate. Due to the NADP+ -dependent
enzyme’s higher abundance, NADPH will be preferentially generated, with the
benefit of an extra proton expelled by transhydrogenase.
Now this is a truly marvelous piece of engineering by Mother Nature, isn’t it?
I’d say this is as close as it gets to intelligent design.
6.14 Is nicotinamide transhydrogenase proof of Life’s creation by intelligent design?
The amount of ATP gained in the respiratory chain for each molecule of glucose
degraded is large, but it cannot be calculated with complete precision and varies
between different physiological states.
17
This futile cycle might also contribute significantly to the proton leak that occurs at high levels
of the proton-motive force (see section 6.10 above).
98 6 The respiratory chain
Total 37.6
Notes: The number of NADH molecules given here includes all molecules accruing
in glycolysis, pyruvate dehydrogenase, and the TCA cycle. The numbers of protons
pumped per molecule of NADH or FADH2 are based on the assumption that com-
plexes I, III, and IV pump 4, 2, and 4 protons, respectively. GTP, which is formed
in the succinate thiokinase reaction in the TCA cycle, is energetically equivalent to
ATP.
6.11.2 Processes other than ATP synthesis that are powered by the proton
gradient
Notes: The amount of ATP given in the preceding slide is a theoretical maximum.
In reality, the ATP yield will be significantly lower, because some protons are
used for purposes other than driving ATP synthase. Most importantly, some
protons are needed for ATP transport. ATP synthesized in the mitochondrion
must be exported to the cytosol, and ADP produced there has to get back in.
This is accomplished by a special transporter protein in the inner mitochondrial
membrane that exchanges ATP and ADP for each other. Since ATP carries one
more negative charge than ADP (ATP4– vs. ADP3– ), this exchange amounts to a net
export of one negative charge, or to the net import of one positive charge per ATP.
Moreover, ionic phosphate produced by cytosolic ATP cleavage also must return
6.12 Answers to practice questions 99
The electrons bypass complex I, which means that fewer protons are extruded from the
mitochondrion compared to electrons that enter the mitochondria via the malate-aspar-
tate shuttle. However, the glycerophosphate shuttle is simpler and probably has higher
capacity than the malate-aspartate shuttle.
6.14: Almost.
Chapter 7
Gluconeogenesis
7.1 Introduction
• The brain requires at least ~50% of its calories in the form of glucose
• Red blood cells exclusively subsist on glucose
• Glucose is a precursor of other sugars needed in the biosynthesis of nucleoti-
des, glycoproteins, and glycolipids
• Glucose is needed to replenish NADPH, which supplies reducing power for
biosynthesis and detoxification
Notes: These considerations make the need for gluconeogenesis quite clear—we
can’t just leave the blood glucose level up to the vagaries of dietary supply.
101
102 7 Gluconeogenesis
Glucose
Glucose-6- P
Fructose-6- P
Fructose-1,6-bis- P
Glyceraldehyde-3- P Glycerol
P -enolpyruvate Acetone
Pyruvate Lactate
Acetyl-CoA
Oxaloacetate α-Ketoglutarate
Fumarate Succinyl-CoA
Amino acids
The major substrate supply for gluconeogenesis is protein, both dietary and en-
dogenous. Protein is first broken down into its constituent amino acids. Those
amino acids that can be converted to pyruvate or any of the TCA cycle inter-
mediates can serve as substrates for gluconeogenesis, and are therefore called
glucogenic.
Leucine, lysine and the aromatic amino acids are degraded to acetyl-CoA or
acetoacetate. Since acetoacetate is a ketone body, and acetyl-CoA can be converted
to ketone bodies, these amino acids are called ketogenic. While it was believed
for a long time that ketogenic amino acids cannot be converted to glucose in
human metabolism, this is not strictly true, since the ketone body acetone can
be converted to pyruvate (see slide 10.4.3). Nevertheless, the contribution of
ketogenic amino acids to glucose regeneration is likely minor.
Gluconeogenesis proceeds only in the liver and the kidneys, and since the liver
is five times larger than the two kidneys combined, it synthesizes most of the
glucose. The pathway does not occur in the brain, fat tissue, or skeletal muscle.
Together with glycogen degradation (see slide 8.3.5), gluconeogenesis ensures
stable blood glucose levels between meals. Gluconeogenesis also enables us to
maintain the necessary glucose levels when on a diet that is rich in protein but low
in carbohydrates.
7.1 Briefly describe the biochemical process of gluconeogenesis.
Most reactions are shared with glycolysis, which we already know, and we here
only need to consider the small number of reactions that are specific to gluconeo-
genesis.
7.2 Reactions in gluconeogenesis 103
The final reaction in glycolysis is the transfer of the phosphate group from
phosphoenolpyruvate (PEP) to ATP. This reaction is irreversible because of the
strongly exergonic nature of the accompanying rearrangement of pyruvate from
the enol to the keto form (see slide 3.3.7). In gluconeogenesis, it takes two en-
zymatic steps to turn pyruvate back into PEP, namely (1) the carboxylation of
pyruvate to oxaloacetate by pyruvate carboxylase, and (2) conversion of oxaloac-
etate to PEP by phosphoenolpyruvate carboxykinase.
O O CO2 O O O
H3 C C C C CH2 C C
OH ATP ADP + Pi HO OH
Notes: With the pyruvate carboxylase reaction, we are able to metabolically fix
CO2 —just like plants! Before we try to claim Kyoto treaty credits for this ability,
however, it is necessary to consider that the very same molecule of CO2 gets
released again in the next step. The whole purpose of transient CO2 fixation is to
enable this subsequent reaction, which is shown in slide 7.2.4.
Arg338 Glu296
HCO–3 Mg2+
Biotin
ADP
Arg292
Notes: The pyruvate carboxylase reaction occurs in two separate steps, which in
human metabolism are carried out in two distinct active sites of a single enzyme
molecule. In E. coli, the two activities are found on separate enzyme molecules. The
first enzyme activity is biotin carboxylase, which attaches CO2 to the coenzyme
biotin.
The figure (rendered from 3G8C.pdb) shows the location of the reactants, as
well as several strategic amino acid residues, within the active site of the E. coli
enzyme. The reaction involves bicarbonate and ATP; the terminal phosphate group
of ATP would fit into the space between ADP, arginine 292, and bicarbonate. The
roles of arginine 338 and glutamate 296 are illustrated in the next slide.
104 7 Gluconeogenesis
O O⊖ O
O OH OH
HN N ⊖O P O HN N HO O P O
OH O⊖ O⊖
R S R S
Notes: Glutamate 296 in the active site initiates the proceedings by deprotonating
bicarbonate, which in turn attacks the terminal phosphate of ATP. This yields
carboxyphosphate, which in turn deprotonates biotin; arginine 338 stabilizes the
anionic biotin intermediate that forms transiently at this stage. The biotin anion
finally attacks the carboxyphosphate, producing phosphate and carboxybiotin.
Figure simplified after a scheme given in [26].
Beyond its role in biotin-dependent carboxylation reactions, carboxyphosphate
(or carbonic-phosphoric anhydride) also occurs as an intermediate in the carbamo-
ylphosphate synthetase reaction, which is the first step in the urea cycle (see slide
12.3.1).
Notes: The second active site—or, in the E. coli pathway, the second enzyme—
transfers the carboxyl group from biotin to pyruvate. The reaction begins with
pyruvate adopting the enoyl configuration. The electrons of the C−
−C double bond
then perform a nucleophilic attack on the carboxyl group, to which biotin readily
yields. The product is oxaloacetate.
7.2 Describe the mechanism of the pyruvate carboxylase reaction.
7.3 Energy balance of gluconeogenesis 105
Notes: In the phosphoenolpyruvate carboxykinase reaction, the CO2 that just had
been attached to the substrate leaves again. This gives rise to an enolpyruvate
anion intermediate that attacks and acquires the terminal phosphate group of
GTP. The product is phosphoenolpyruvate, which is an intermediate of glycolysis.
All reactions between phosphoenolpyruvate and fructose-1,6-bisphosphate are
reversible; we can therefore skip ahead to the latter metabolite.
Fructose-1,6-bisphosphate + H2 O Fructose-6-phosphate + Pi
Glucose-6-phosphate + H2 O Glucose + Pi
Notes: These reactions revert the substrate phosphorylations that occur in the
first and the third step of glycolysis, which are catalyzed by hexokinase and phos-
phofructokinase, respectively. In gluconeogenesis, the phosphate groups are sim-
ply hydrolyzed off, which is not a very difficult sort of reaction.
7.3 Which enzymes in glycolysis catalyze irreversible reactions, and how are these reac-
tions bypassed in gluconeogenesis?
2 pyruvate 2 oxaloacetate 2
2 oxaloacetate 2 PEP 2
2 3- P -glycerate 2 1,3-bis- P -glycerate 2
Total 6
As pointed out above (section 7.1), substrate carbon for gluconeogenesis accrues
mostly from amino acid degradation and is harvested at the level of pyruvate or
of TCA cycle intermediates. Pyruvate carboxylase, which turns pyruvate into a
TCA cycle intermediate, is important not only in gluconeogenesis, but also in the
replenishment of TCA cycle intermediates, which may become depleted through
diversion to the biosynthesis of amino acids or of heme. Therefore, this enzyme is
expressed not only in the organs that perform gluconeogenesis (liver and kidneys)
but ubiquitously.
Gluconeogenesis is also part of two interorgan cycles, namely, the Cori cycle
and the glucose-alanine cycle. These will be discussed after several other partici-
pating pathways have been introduced (see slides 8.5.3 and 12.3.5).
Cytosol Mitochondria
H+ H+
pyruvate pyruvate
oxaloacetate oxaloacetate
+
NADH+H NADH+H+ malate
dehydrogenase
NAD+ NAD+
malate malate
dicarboxylate
transporter
Pi Pi
ATP ATP
Notes: In keeping with its role in replenishing TCA cycle intermediates, pyruvate
carboxylase resides inside the mitochondria. The next step in gluconeogenesis,
catalyzed by phosphoenolpyruvate carboxykinase, occurs in the cytosol; therefore,
oxaloacetate must in some way be exported from the mitochondria again.
We had already seen that the mitochondrial concentration of oxaloacetate is
low, and that the malate dehydrogenase equilibrium favors malate (section 5.5).
It turns out that substrate export occurs indeed at the level of malate, which
is exchanged for phosphate by a mitochondrial transport protein known as the
dicarboxylate carrier. The malate dehydrogenase reaction is then reversed in the
7.4 Interactions of gluconeogenesis with other pathways 107
cytosol; the NADH produced can be used in the reversal of the glyceraldehyde-3-
dehydrogenase reaction (see slide 3.3.5), which occurs later on in gluconeogenesis.
The phosphate that entered the mitochondrion in exchange for malate can
be used by ATP synthase, and the ATP be exchanged for cytosolic ADP, which
balances the entire transport cycle and supplies one ATP to the cytosol, where it
may for example be used by phosphoglycerate kinase in gluconeogenesis.
The mitochondrial export of malate is susceptible to inhibition by methyl-
malonate. The coenzyme A thioester of methylmalonate occurs in the metabolic
utilization of fatty acids with uneven numbers of carbon atoms (see slide 10.3.6).
This pathway requires vitamin B12 . A lack of the vitamin will cause free methyl-
malonate to accumulate. This may inhibit gluconeogenesis and thus account for
the clinical hypoglycemia that sometimes accompanies vitamin B12 deficiency [27].
7.4 Explain how oxaloacetate is transported from the mitochondrion to the cytosol to
supply gluconeogenesis.
NAD+
acetaldehyde
NADH+H+
acetyl-CoA glucose
Notes: Like gluconeogenesis, ethanol degradation occurs in the liver. The utiliza-
tion of one molecule of ethanol by alcohol dehydrogenase and then aldehyde
dehydrogenase yields acetate, which is converted to acetyl-CoA by acetate thioki-
nase.
For each molecule of ethanol degraded, two equivalents of NAD+ are reduced
to NADH. This raises the cytosolic [NADH]/[NAD+ ] ratio, which in turn reduces both
pyruvate and oxaloacetate and thus deprives gluconeogenesis of its substrates. In
alcoholic patients, this problem is often compounded by a low intake of carbohyd-
rates. Clinically manifest hypoglycemia with unconsciousness is a well-known and
potentially dangerous complication in alcohol addiction.
7.5 Explain how alcohol degradation interferes with gluconeogenesis.
108 7 Gluconeogenesis
glucose fructose-6- P
Pi ATP Pi ATP
H2 O ADP H2 O ADP
glucose-6- P fructose-1,6-bis- P
oxaloacetate PEP
ADP+Pi ADP
Notes: Each of these short cycles combines one enzyme from glycolysis (blue
arrows) with one or two enzymes from gluconeogenesis (red arrows). In each case,
the net result of the cycle is simply the consumption of ATP or GTP and the release
of heat.
These cycles have been shown to run in living cells at appreciable levels, possi-
bly for the sake of heat production; and as pointed out in chapter 2, such substrate
cycles also sharpen up regulatory responses (see slide 2.5.7). Nevertheless, their
activity must be kept in check in order to avoid excessive wastage of ATP. Some,
but not all of the regulatory mechanisms that exercise this control are understood.
Glucose Glucose
Pi ATP
Glucose-6-
phosphatase Hexokinase
H2 O ADP+Pi
Glucose-6- P Glucose-6- P
to the ER. Interestingly, the transporter and the phosphatase are expressed in
many tissues, not just those that perform gluconeogenesis [28]; it is not clear at
present what, if any, function other than futile cycling this might serve.
Pi Fructose-6- P ATP
ATP
Fructose-1,6-
AMP Phosphofructokinase
bisphosphatase
Fructose-2,6-bis- P
H2 O Fructose-1,6-bis- P ADP
Protein kinase A
Fructose-2,6-bis- P
Fructose-6- P
110 7 Gluconeogenesis
7.6 Explain the role of fructose-2,6-bisphosphate in the regulation of glycolysis and glu-
coneogenesis.
N O−
N
O P O− O
O N N
O O O P O−
O
HO O−
CH2 OH
O P O OH
OH
O−
Notes: This slide just shows the structures of the secondary messengers intro-
duced in the previous one. In the structure of fructose-2,6-bisphosphate, the
phosphate group attached to the 2′-position is highlighted.
It should be noted that, among the three allosteric effectors of phosphofructok-
inase and fructose-1,6-bisphosphatase, ATP and AMP reflect the energetic situation
of the cell itself, whereas the level of fructose-2,6-bisphosphate is regulated by
hormonal stimulation and thus controls the same enzymes on behalf of the needs
7.6 Answers to practice questions 111
of the body as a whole. This is a nice example of how regulatory signals of different
origin and meaning are integrated at the molecular level.
Glycogen metabolism
8.1 Overview
n
pV = nRT ⇐⇒ p = RT
V
• Glycogen amounts to 10% of the liver’s wet weight, equivalent to 600 mM
glucose
• When free, 600 mM glucose would triple the osmotic activity of the cytosol—
liver cells would swell and burst
113
114 8 Glycogen metabolism
8.2.3 The size of glycogen particles is limited by crowding in the outer layers
Notes: According to the rules detailed in the preceding slide, the number of
branches will double with each successive generational layer of the glycogen mo-
lecule. However, the amount of space available to those branches will only grow
in proportion to the square of the particle radius or, approximately, the square
of the number of generations. Taking into account the actual dimensions and
architecture of the polymer, it has been calculated that in the 13th generation the
required space would exceed the available space. Therefore, a glycogen molecule
can contain no more than 12 generational layers. This implies that a single glyco-
8.2 Glycogen structure 115
gen molecule can contain up to approximately 54,000 glucose residues; it will have
a molecular weight of almost 107 Da and a diameter of approximately 25 nm [29].
available
required
Layer volume
0 5 10 15
Layer generation
Electron microscopy shows glycogen particles whose dimensions agree well with
this theoretical maximum size of single molecules; these are referred to as β
particles. In many tissues, variable numbers of β particles are found clustered
into so-called α particles. Interestingly, α particles can be broken up with thiol-
reducing agents, which implies that they are held together by disulfide bonds.
Disulfides usually form between protein molecules. In addition to such scaffolding
proteins, glycogen particles also contain a considerable number of enzymes and
regulatory proteins [30], some of which will be discussed below.
8.2.4 Glycogen is more loosely packed and more soluble than amylose
Glycogen Amylose
Notes: The above structural model of glycogen assumes a relatively loose packing
of the glucose residues within the α-1 4-linked linear stretches. However, per-
fectly linear polyglucose—that is, amylose—adopts a much more densely packed
helical structure. In this structure, more hydroxyl groups are engaged in hydrogen
116 8 Glycogen metabolism
bonds with other glucose residues rather than with water; amylose therefore has
low aqueous solubility.1
Unlike amylose, which mostly serves for long-term storage in plant bulbs and
seeds, glycogen typically is degraded within hours of synthesis; this rapid turnover
is facilitated by its loose structure. However, if branch formation breaks down,
aberrant, condensed glycogen particles may arise that are no longer amenable to
regular turnover. Various enzyme defects that interfere with branch formation
cause the accumulation of such particles inside the cells; an example is the defect
of the enzyme laforin in Lafora disease (see slide 8.6.4).
Aberrant glycogen particles also arise spontaneously in normal metabolism.
The high density of polyglucose chains in the outermost layers of the glycogen
molecule may interfere with the activity of branching enzyme and may promote
tighter packing. Lysosomal glycogen degradation (see section 8.3.7) may have
evolved as a pathway to dispose of such dysfunctional particles.2
Notes: Both glycogenin and glycogen synthase use an activated form of glucose,
UDP-glucose, which is formed from glucose-6-phosphate in two steps. Phospho-
glucomutase first transforms glucose-6-phosphate to glucose-1-phosphate (1),
1
Cooking starch has the effect of breaking up hydrogen bonds within amylose and increase its
degree of hydration. Amylose thereby becomes digestible by amylase. Cooking food ranks among
mankind’s greatest inventions, almost comparable to Twitter and the iPad.
2
Undegraded insoluble glycogen particles may be seen inside the cells of aged individuals. Such
particles are described as corpora amylacea in the brain and as cardiac colloid in heart muscle.
8.3 Glycogen synthesis and degradation 117
which is then converted to UDP-glucose (2). The latter reaction requires uridine
triphosphate (UTP) and releases pyrophosphate.3
O− Glucose-1-phosphate
O P O−
O CH2 OH
O 1 O
OH OH O
HO OH HO O P O− UTP
OH OH O− O
Glucose-6-phosphate NH
O− O− O−
−O P O P O P O N O
O
O O O
O OH OH
CH2 OH
2
O NH
OH
O− O− O− O−
HO O P O P O N O −O P O P O−
O
OH
O O O O
UDP-glucose OH OH Pyrophosphate
The UDP-glucose that is used in the Leloir pathway of galactose degradation (slide
4.3.1) is derived in the same manner. UDP-glucose is also the precursor of UDP-
glucuronic acid, which is used in the conjugation of bilirubin (section 17.4) and of
xenobiotics (section 18.3).
8.2 Explain how UDP-glucose is formed, and name its metabolic destinations.
Notes: Glycogenin is a small bifunctional protein that serves both as the starter
substrate and the polymerase that synthesizes the initial linear strand of glucose
residues. It begins the synthesis by attaching the initial glucose residue to a
strategic tyrosine side chain of itself, and then successively adds several more
glucose residues to the sugar’s 4′ end.
The linear chain synthesized by glycogenin may contain up to ~12 glucose
residues. This chain length would be long enough to serve as a substrate for
branching enzyme, and it seems likely that the next step is indeed the introduction
of the first branch. Alternatively, it is possible that the free end is first extended
some more by glycogen synthase before branching occurs. In any event, after
branching has occurred, glycogen synthase extends both of the two available 4′
ends. The remainder of the molecule is built through the alternating actions of
3
Wherever pyrophosphate is released, it is subsequently cleaved to two phosphate ions by py-
rophosphatase. This cleavage is strongly exergonic and keeps the concentration of pyrophosphate
very low, which in turn makes its release more exergonic. The release of pyrophosphate therefore
provides a stronger push to a reaction than the release of monophosphate.
118 8 Glycogen metabolism
branching enzyme and glycogen synthase; the cycle repeats until the glycogen
reaches its inherent size limit at approximately 10 MDa (see slide 8.2.3).
... HO Gg
Branching enzyme
Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc O Gg
UDP-Glc
UDP
UDP-Glc
The reactions performed by glycogenin itself and by glycogen synthase are equiv-
alent. It is interesting to note that the carbon 1 of each glucose subunit is in the
α-configuration both in UDP-glucose and in glycogen. What does this tell us about
the mechanism of the reaction?
Glu Glu
CH2 OH CH2 OH
⊖O O O O
O O
OH OH CH2 OH CH2 OH
HO O O
HO OH OH
HO HO
O ⊖O O
UDP
...
O P O− O OH OH
O
NH
O P O−
O N O
O CH2 OH CH2 OH CH2 OH
O O O
OH OH OH
OH OH HO O O ...
OH OH OH
O
OH
HO
CH2 OH HO
CH2 OH CH2 OH
+
Oδ O O O
OH δ+ OH OH
O P O− O
HO O O ...
O
HO H OH OH NH
O P O−
O⊖
O N O
O P O− O O
O
NH
O P O−
OH OH
X N O
O
Pi
Glc-1- P Pi
phosphorylase Glc Glc Glc Glc Glc Glc Glc Glc Glc O Gg
Glc-1- P
debranching enzyme
H2 O Glc
Glc
Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc O Gg
In the liver, which stores glycogen for the benefit of the entire body, the lion’s share
of glucose-6-phosphate will be dephosphorylated by glucose-6-phosphatase and
then released into the circulation; overall, this is equivalent to outright hydrolysis.
However, muscle uses glycogen largely toward its own energy needs, and therefore
glucose-6-phosphate will usually be funneled straight into glycolysis. In this case,
the use of phosphorolysis instead of hydrolysis bypasses the hexokinase reaction,
thereby saving one equivalent of ATP.5
Glycogen phosphorylase only degrades the chain ends to within four residues
of a branching point. Then, debranching enzyme takes over and transplants
the stub to another free end, where it becomes again a substrate for phosphory-
lase. However, this reaction leaves behind a single residue attached by a α-1 6-
glycosidic bond. This residue is subsequently released by the same enzyme as free
glucose through hydrolysis.
8.3 Give a summary of glycogen synthesis and degradation.
OH OH OH
HO δ+ HO
O HO O O
δ+
HO HO
HO
OH H OH
OH O−
O O
Glycogen O P
H Glycogen O−
O O
−O
O −O P O−
P
O O− O
H
H H
O− O
O O −O −O
P O P O
P
−O
O O O
N N N
N N N
OH OH OH
Glycogen
AMP
Glucose-6- P
UDP-glucose
Glucose-1- P
Protein kinase A
Glycogen Glycogen
synthase synthase- P
Phosphorylase Phosphorylase
kinase kinase- P
Phosphorylase Phosphorylase- P
Inhibition by glucose + −
2+
Activation by Ca − +
Activation by AMP even when − +
unphosphorylated
As stated above, the two tissues that have the most significant pools of glycogen
are the liver and skeletal muscle. Liver glycogen is turned over rapidly; it serves
as the major reserve of blood glucose during short-term fasts. Once liver glycogen
is depleted, muscle glycogen can be drawn down; this, however, requires some
roundabout metabolic trickery.
Notes: The liver mobilizes glucose from its glycogen store via glycogen phospho-
rylase and phosphoglucomutase, which yields glucose-6-phosphate. The latter is
transported to the endoplasmic reticulum, where it is dephosphorylated. Glucose
is taken back to the cytosol and released into the bloodstream.
124 8 Glycogen metabolism
Glycogen
Glucose-1- P
endoplasmic reticulum
Glucose-6- P Glucose-6- P
glucose-6-phosphatase glucokinase
Glucose Glucose
cytosol
extracellular space
Glucose
Some of the glucose will be rephosphorylated before making it out of the cell,
creating the futile cycle discussed in slide 7.5.2. However, the dominant glucose
phosphorylating enzyme in the liver is glucokinase, which has fairly low affinity for
glucose (see slide 3.5.3); therefore, enough glucose will escape rephosphorylation
and be released into the bloodstream.
Glycogen
Glucose-1- P
endoplasmic reticulum
Glucose Lactate
Notes: Muscle glycogen primarily serves the energy needs of muscle tissue itself;
during prolonged physical exercise, most of it is broken down to glucose-6-phos-
phate and then consumed via the usual pathways right within the cells that stored
it. As discussed above, this usage is facilitated by calcium-mediated activation of
glycogen phosphorylase.
Under suitable conditions, namely, prolonged fast without physical exercise,
muscle glycogen can also contribute to the replenishment of blood glucose. How-
ever, even though muscle cells have been shown to express glucose-6-phosphatase
8.5 Interorgan relationships in glycogen metabolism 125
[32] and thus are, in principle, able to produce free glucose, they should find it dif-
ficult to release it. This is because muscle contains hexokinase, which has a much
greater substrate affinity than glucokinase and therefore will keep the intracellular
level of free glucose well below the extracellular concentration. The net transport
of glucose should therefore be directed inward at all times; this agrees with all the
physiological evidence that I could find.
The way around this obstacle is to convert glucose-6-phosphate to pyruvate
and then lactate. At a low rate, lactate formation occurs even in resting muscle
and under aerobic conditions. This lactate is derived in various proportions from
blood glucose and glycogen, respectively. In animal experiments, epinephrine
promotes glycogen utilization and lactate release [33], but overall the hormonal
control of this process and the magnitude of its contribution to systemic glucose
control are not well characterized.
8.7 Explain how substrate carbon derived from muscle glycogen may be made available
to bolster blood glucose levels.
Glucose Glucose
Notes: While skeletal muscle relies on oxidative metabolism most of the time,
some other tissues, notably red blood cells and lymphocytes, which collectively
account for some 4 kg of cell mass, depend mostly or even exclusively on anaerobic
glycolysis even under aerobic conditions. The lactate released in peripheral tissues
is scooped up by the liver, which converts it back to glucose through gluconeogen-
esis. This process is known as the Cori cycle, named after its discoverers Carl and
Gerti Cori, who worked it out as early as 1929 [34].
Skeletal produces lactate at a very much higher rate during short bouts of
maximal exercise when the ATP demand exceeds the capacity for aerobic metabo-
lism. Some textbooks state that the Cori cycle resupplies the muscle with glucose
in this situation also. This is, however, quite impossible. During intense exercise,
the cardiac blood output is diverted from the visceral organs to skeletal muscle.
Therefore, when ATP demand exceeds the oxygen supply of skeletal muscle, the
oxygen shortfall would be even greater in the liver, should it indeed attempt to
make enough ATP for sustaining the muscle through gluconeogenesis; and even
126 8 Glycogen metabolism
with sufficient oxygen, its capacity for making glucose would fall far short of the
muscles’ voracious appetite.
Anaerobic exercise can be sustained for only short periods of time anyway.
During this period, the lactate turned out by skeletal muscle will simply accumu-
late; it will then slowly be scooped up by the liver and turned back into glucose
after we have collapsed at the side of the track to catch our breath.
8.8 Describe the Cori cycle.
Genetic defects have been described for several enzymes of glycogen metabolism.
The clinical syndromes associated with these defects are referred to as glycogen
storage diseases. While these conditions are not particularly common, they do
shed some light on the physiological significance of glycogen metabolism. Some
conditions are clinically severe and are the focus of ongoing therapeutic research.
A few examples are briefly discussed below.
phosphate. This impedes the regeneration of ATP and raises the level of AMP,
some of which then enters degradation to uric acid [35].6
The clinical severity of this disease may vary, presumably due to different
levels of residual enzyme activity. Some cases may be managed with a diet of
frequent, starch-rich meals, which helps to avoid hypoglycemia. In more severe
cases, liver transplantation may become necessary.
8.9 Name the enzyme defect that underlies von Gierke disease, and explain why it gives
rise to hypoglycemia.
Normal skeletal muscle Glycogen aggregates Infant chest X-ray, Infant with Pompe
(transverse section) in Pompe disease normal heart disease, distended heart
6
This mechanism bears some resemblance to the hyperuricemia that can be triggered in healthy
patients through fructose overload (see slide 16.6.4).
7
Maltose is a suitable model substrate, but the real role of this “maltase” enzyme is to cleave the
α 4-glycosidic bonds in glycogen.
128 8 Glycogen metabolism
Notes: Since liver and muscle phosphorylase are distinct isozymes, defects usually
affect one and spare the other.8 In McArdle’s disease, the muscle isoform is
selectively affected. An unexplained symptom in this disease is the so-called
“second wind” phenomenon: during physical activity, patients initially fatigue
rapidly, but then recover to a degree under continued exercise.
The lack of muscle phosphorylase should also inhibit the utilization of muscle
glycogen toward blood glucose stabilization by way of the Cori cycle (see slide
8.5.3); one might therefore expect that McArdle’s disease might involve episodes
of hypoglycemia. Interestingly, however, the literature does not contain reports of
hypoglycemia in these patients.
activates protein kinase A, which directly phosphorylates and thereby inactivates glycogen
synthase. The kinase also phosphorylates a secondary kinase that phosphorylates, and
thereby activates, glycogen phosphorylase.
8.6: Glycogen is a polymeric form of glucose that allows storage of the latter in large
amounts. The highest concentrations are found in the liver. Liver, and possible muscle
cells, store glucose as glycogen under the influence of insulin, and they degrade glycogen
to glucose and release it into the circulation under the influence of glucagon and epineph-
rine. The synthesis and degradation of glycogen helps to maintain a stable blood glucose
concentration through phases of varying supply and demand.
8.7: Skeletal muscle contains all enzymes for converting glycogen to glucose, but due
to its content of hexokinase is likely unable to accumulate enough free glucose to effect
its release into the circulation. However, glycogen-derived glucose may be converted to
pyruvate and then lactate, which may be released and enter gluconeogenesis in the liver.
8.8: The Cori cycle is an interorgan cycle, combining gluconeogenesis in the liver with
anaerobic glycolysis in peripheral tissues. It enables the disposal and utilization of lactate
formed in anaerobic glycolysis.
8.9: The deficient enzyme is glucose-6-phosphatase. Without it, the liver and kidney are
unable to release free glucose formed through glycogen degradation and gluconeogen-
esis, which are the key pathways for blood glucose stabilization—that is, prevention of
hypoglycemia—in fasting periods.
Chapter 9
2 NADP+
Ribose-5- P
2 NADPH+H+ CO2
Notes: The previous chapters have shown that glucose-6-phosphate has a central
place in carbohydrate metabolism. This chapter describes yet another pathway
that starts with this key metabolite, namely, the hexose monophosphate shunt,
or HMS for short. Since this pathway comprises both pentoses and hexoses, it
is sometimes also referred to as the pentose phosphate pathway. It serves two
major functions that are important for biosynthesis, namely (1) the regeneration
of NADPH from NADP+ , and (2) the provision or utilization of ribose. Within the
pathway, we can distinguish two phases:
1. the oxidative phase, in which glucose-6-phosphate is oxidized and decarboxy-
lated to ribulose-5-phosphate, and which yields two equivalents of NADPH,
and
131
132 9 The hexose monophosphate shunt
OH OH
O + + O
CO2 NADPH+H NADP
OH OH OH
O O HO
3 3
OH OH OH
OH O OH O OH O
O P O− O P O− O P O−
O− O− O−
Ribulose-5- P 6- P -Gluconate
Notes: The sugar shuffle stage involves the enzymes ribulose-5-phosphate epimer-
ase (RE), ribulose-5-phosphate isomerase (RI), transketolase (TK), and transaldolase
(TA). They bring about the following reactions:
1. Two molecules of ribulose-5-phosphate are converted to xylulose-5-phosphate
by ribulose-5-phosphate epimerase, and a third one is converted to ribose-
5-phosphate by ribulose-5-phosphate isomerase.
2. Transketolase transfers a C2 unit from one xylulose-5-phosphate to the ribose-
5-phosphate, yielding glyceraldehyde-3-phosphate and the C7 sugar sedohep-
tulose-7-phosphate.
3. Transaldolase transfers a C3 unit from sedoheptulose-7-phosphate back to
glyceraldehyde-3-phosphate, which yields fructose-6-phosphate and the C4
sugar erythrose-4-phosphate.
4. Transketolase transfers a C2 unit from the second molecule of xylulose-5-phos-
phate to erythrose-4-phosphate. This yields a second molecule of fructose-6-
phosphate and again glyceraldehyde-3-phosphate.
At the conclusion of the reactions depicted in this slide, three molecules of
ribulose-5-phosphate have been converted to two molecules of fructose-6-phos-
phate and one molecule of glyceraldehyde-3-phosphate. Fructose-6-phosphate can
134 9 The hexose monophosphate shunt
TK
OH
O OH
OH RI O HO O
O OH OH O HO
OH OH OH OH OH
OH OH OH OH OH
O P O P O P O P O P
OH RE OH TK
O O
OH HO O
OH OH OH
O P O P O P
Notes: The shuffling of sugars differing in chain length is brought about by just
two enzymes, namely, transaldolase and transketolase. While the sugar substrates
they act upon may appear to be quite varied at first glance, they fall into just two
homologous classes, within each of which the members differ only in the number
of CHOH groups in their “tails”. The two enzymes only interact with the “head”
parts of each of these sugar molecules, so that the chain length of the remainder
doesn’t enter into the picture and does not create a need for separate enzyme
specificities.
The basic idea of chain length variation is that transketolase always transfers
two-carbon units, whereas transaldolase always transfers three-carbon units. A cu-
riosity that results from this modus operandi is the occurrence of an eight-carbon
sugar [38]. This molecule does not have a role in the pathway, nor does it serve any
9.2 Reactions in the hexose monophosphate shunt 135
OH OH OH
TK TK TK
O O TA O TA
HO HO HO
OH OH OH
OH P
O OH OH
O RE
O P OH
OH P
O
OH
O
O P RI OH O
OH OH O
Ribulose-5- P
OH OH OH
O P O P O P
9.2 Imagine three players who hold five chips each, who have been asked to trade chips
in amounts of no less than two and no more than three, so that they will end up with six,
six and three chips, respectively. How can they do this with the smallest possible number
of trades?
OH S OH
O H⊕ ⊕ OH
N
HO O R
R
OH H OH S OH
R O P ⊕
O P ⊖ OH
N
S
R
⊕ ⊖
N
R
R
O
S OH
OH OH
⊕ O H H⊕
N O P
O
R HO O
HO
OH OH
OH
OH OH
OH
OH OH
OH
O P O P
O P
OH OH OH
Lys OH– Lys
⊕
Lys
NH2 O N N
H H
HO HO HO
OH O H
OH OH
OH OH
O P O P O
OH
OH
OH OH O P
⊕
Lys NH2 O Lys N
H
HO HO H⊕
OH–
OH OH O
OH OH OH
O P O P O P
Notes: Transaldolase also forms a covalent intermediate with the fragment of the
sugar molecule it transfers, and once more the carbonyl bond serves as the point
of attack for cleavage. However, transaldolase cleaves the bond after the second
carbon atom, resulting in the transfer of a C3 unit. The two stages of the reaction
are again reversals of each other, with the exception that the two sugar substrates
differ in chain length.
Considering their mechanisms, it is clear that both the transketolase and
the transaldolase reactions are readily reversible. So are the isomerase reactions
that interconvert the various pentose phosphates. The entire pathway therefore
can proceed in either direction and bring about the interconversion of pentoses,
hexoses, and sugars of other lengths, in any amounts and proportions as needed.
9.3 Discuss similarities and differences between the reaction mechanisms of transaldolase
and transketolase.
Our discussion of the HMS pathway is now complete, and we will now have a look
at the various metabolic functions of NADPH.
9.3 The physiological role of NADPH 137
H H O H H O
NH2 NH2
N N
O O O O
−O P O OH OH −O P O OH OH
NH2 NH2
O O
−O P O N −O P O N
N N
O N N O N N
O O
OH OH OH O
−O P O
O−
Notes: Why is NADPH needed in addition to NADH? The two coenzymes only differ
by one phosphate group, and that group is far away from where the action is: The
redox-active group is the pyridine ring in the nicotinamide moiety (highlighted),
whereas the extra phosphate in NADP is located on the adenosine moiety at the
other end of the molecule.
While the phosphate group does not make any difference to the redox chem-
istry performed by the two coenzymes,1 it enables them to interact with separate
sets of enzymes. Consider that all enzymes which consume or regenerate NAD+
will share the same pool of the cosubstrate, and the reaction equilibria of all of
+
them will be affected by the same ratio of oxidized to reduced form, [NAD ]/[NADH].
The extra phosphate group on NADP allows it to interact with another, differ-
ent set of enzymes. Therefore, because the coenzymes participate in separate sets
of equilibria, they can themselves be maintained in different redox states. To use a
simile: the two coenzymes are like two different currencies—both are money, but
it is possible to tune the cost of borrowing of each separately to different economic
objectives. Inside the cell, NAD is mostly oxidized. The ready availability of NAD+
will help to speed up the oxidative reactions in the TCA cycle and glycolysis. In
contrast, NADP is mainly found in the reduced state, which will promote reductive
reactions in biosynthesis.
The choice of of either NAD or NADP as the cosubstrate will not only affect
the turnover rate of a redox reaction but also its free energy (∆G). According to
[39], the [NADH]/[NAD+ ] ratio in the cytosol is 0.001, while the [NADPH]/[NADP+ ] ratio is
100. Neglecting the very slight difference in ∆G0 , the 105 times higher relative
abundance of NADPH works out to a difference of ~30 kJ/mol in actual ∆G. This
amount of energy is similar to that released by the hydrolysis of ATP to ADP, which
is not a coincidence (see next slide).
1
If we use the same concentrations of reduced and oxidized forms in vitro, the difference in their
redox potentials is very small; that is, both have virtually the same standard redox potentials.
138 9 The hexose monophosphate shunt
Cytosol Mitochondria
H+ H+
pyruvate pyruvate
oxaloacetate
malic
NADP+ enzyme NADH+H+ malate
dehydrogenase
NAD+
malate malate
dicarboxylate
transporter
Pi Pi
Notes: While the HMS is the major source of NADPH in most tissues, some other
pathways contribute to the supply, particularly in tissues that synthesize fatty
acids or sterols.
The shuttle shown here reuses most elements of the one that moves oxaloac-
etate from the mitochondria to the cytosol for gluconeogenesis (see slide 7.4.1,
which also shows how to balance the transport of phosphate). However, in the final
step of the shuttle considered here, cytosolic malate is not converted to oxaloac-
etate but is instead decarboxylated by malic enzyme, which generates NADPH; the
pyruvate that is left over can reenter the mitochondria.
On the mitochondrial side, the shuttle consumes one equivalent of NADH and
ATP each. The ATP drives the carboxylation of pyruvate, which is reverted by malic
enzyme. Assuming that malic enzyme can recover the full free energy acquired
from ATP during carboxylation and apply it toward the reduction of NADP+ , the
enzyme should indeed be able to raise the ratio of cytosolic NADPH to NADP+
to beyond that of NAD by the factor of 105 explicated in slide 9.3.1. While this
assumption may be a bit optimistic, it is also not strictly necessary, since the
[NADH]/[NAD+ ] ratio is significantly higher in the mitochondria than in the cytosol,
which helps push the cycle in the indicated direction. Overall, the participation
of ATP in this cycle accounts for the difference in concentration and free energy
between NADPH and NADH discussed in the preceding slide.
NADH+H+ NAD+
nicotinamide nucleotide
H+
transhydrogenase
isocitrate
CO2 CO2
dehydrogenase
citrate/isocitrate
carrier
malate malate
oxoglutarate
carrier
Note that this cycle does not involve the hydrolysis of ATP. The single proton
imported by the transhydrogenase does not provide quite the same amount of free
energy as ATP, and the question therefore arises how sufficient driving force for
the overall process is derived. As far as I can see, the only other contributing factor
is the more reduced state of NAD inside the mitochondrion as compared to the
cytosol.2 It may thus be that this shuttle works only when both the proton-motive
force and the mitochondrial NADH are at high levels, while cytosolic NADPH is
lowered due to high rates of consumption, such as for example during fatty acid
synthesis in fat tissue [40].
9.4 Explain how cytosolic NADPH can be regenerated.
2
The exchange of isocitrate for α-ketoglutarate is electroneutral, and thus does not derive addi-
tional drive from the proton-motive force.
140 9 The hexose monophosphate shunt
N N NH
O2 H2 O O2 H2 O
H2 N NH2 H2 N NH H2 N O
+
OH
N−
−O
Notes: Nitric oxide produced by NOS diffuses out of the cell of origin, for example
a vascular endothelial cell, and then into another one, such as a vascular smooth
muscle cell. Within its target cell, NO binds and activates soluble guanylate cyclase
(sGC), which then begins to make cyclic GMP (cGMP). Like cAMP, cGMP acts as a
second messenger inside the cell.
Also like cAMP, cGMP targets multiple effector molecules. Activation of cGMP-
dependent protein kinase (cGK) results in the phosphorylation of various proteins.
In vascular smooth muscle, this induces relaxation, which in turn lowers the blood
pressure; this is exploited by NO-releasing drugs in the treatment of hypertension.
Phosphodiesterase 5 (PDE) is activated by cGMP, too, and begins to degrade both
9.3 The physiological role of NADPH 141
cAMP and cGMP. Actuation of cyclic nucleotide-gated cation channels affects the
membrane potential and the cellular calcium level.
NADPH oxidase
2O2 + NADPH 2O–2 + NADP+ + H+
Superoxide dismutase
2O–2 + 2H+ H 2 O2 + O 2
Myeloperoxidase
H2 O2 + Cl– OCl– + H2 O
Notes: Where not needed for immune defense, reactive oxygen species are harm-
ful rather than useful; for example, they can react with non-saturated fatty acyl
residues in lipid membranes (see below). Nevertheless, some ROS continually form
as byproducts of respiration and also of oxygen transport in erythrocytes, since
binding to hemoglobin offers O2 an opportunity to steal an electron from the heme
and turn itself into superoxide.
ROS toxicity is controlled by glutathione (G-SH), which is present in the cells
at low millimolar concentrations. An important step in the detoxification of ROS
is the reduction of hydrogen peroxide (H2 O2 ) to water by glutathione peroxidase
142 9 The hexose monophosphate shunt
(1). In the process, glutathione is oxidized to its disulfide form (G-SS-G). Reduced
glutathione is then regenerated at the expense of NADPH by glutathione reductase
(2).
H2 O2 2H2 O
HO OH HO OH
O O 1 O O
NH2 NH2
NH NH
O O
O O
HN S H H S NH HN S S NH
O O
O O
HN HN
H2 N H2 N
O O 2 O O
HO OH HO OH
+ +
NADP NADPH+H
2 G−SH G−SS−G
O O
HO
NH NH
H2 N N O H2 N N O
H H
Divicine Isouramil
Notes: The word “favism” derives from the Latin name of the broad bean, Vicia
faba. Broad beans contain several pyrimidine derivatives such as isouramil and
divicine that can catalyze the formation of ROS through redox cycling, which is
explained in the next slide.
O 2 H2 O GSSG NADPH
O
NH 3 4
HN N O
NADP+ 2 GSH H H2 O2 2 GSH NADP+
4 1 2
O
NADPH GSSG O2
HO
NH
H2 N N O
H
Notes: This scheme outlines the redox cycle induced by isouramil. Reactions (1)
and (2) occur spontaneously, without any need for enzymatic catalysis. The H2 O2
formed in reaction (2) is reduced by glutathione peroxidase (3). The glutathione
disulfide formed in reactions (1) and (3) is reduced at the expense of NADPH in
reaction (4) by glutathione reductase. Therefore, this redox cycle consumes four
equivalents of reduced glutathione (GSH) in every full turn. Divicine and some
other substances contained in broad beans cause analogous cycles.
Notes: Malaria parasites multiply inside erythrocytes, where they feed on hemo-
globin. They digest the protein (globin) but leave behind the heme, which is toxic to
them because it catalyzes the formation of reactive oxygen species. To reduce the
toxicity of heme, the parasites induce its crystallization; the crystalline deposits
are visible inside the infected cells as brownish so-called malaria pigment. The
parasites also produce several heme-binding proteins to aid in the detoxification.
144 9 The hexose monophosphate shunt
Another factor that may contribute to the protection of the parasites from heme
toxicity is the red cells’ own glutathione-dependent ROS scavenging pathway. This
provides a plausible explanation for the observation that glucose-6-phosphate
dehydrogenase deficiency affords partial protection from malaria, which has led to
the enrichment of this enzyme defect in the human gene pool in endemic malaria
areas.
O2 H2 O2
Primaquine
OH O
O O O
CYP450
N N N
NH NH N
H2 N H2 N H2 N
GSSG 2 GSH
lysyl residue with transaldolase) and in the position of bond cleavage/formation relative
to the carbonyl groups.
9.4: Aside from the hexose monophosphate shunt, cytosolic NADPH can be regenerated
using mitochondrial reducing power. In one such pathway, mitochondrial malate is translo-
cated to the cytosol and converted to pyruvate by malic enzyme, which yields NADPH; the
pyruvate can be converted back to malate by pyruvate carboxylase and malate dehydroge-
nase in the mitochondria.
Another pathway involves isocitrate, for which there is an NADP-dependent dehy-
drogenase both in the cytosol and the mitochondria. A cycle propelled by nicotinamide
nucleotide transhydrogenase effects the formation of isocitrate in the mitochondria and
its dehydrogenation in the cytosol.
9.5: See list in slide 9.3.4.
9.6: NADPH is used by NADPH oxidase to reduce O2 to superoxide, which can then give
rise to H2 O2 and HOCl through the actions of superoxide dismutase and myeloperoxidase.
Superoxide can react with NO, whose formation by nitric oxide synthase also requires
NADPH, to peroxynitrite. All these molecules have antimicrobial activity.
9.7: Favism is caused by a genetic defect of glucose-6-phosphate dehydrogenase. This
defect limits the capacity of erythrocytes to regenerate reduced glutathione.
Broad beans contain isouramil and several similar compounds that cause redox cycles.
In such a redox cycle, molecular oxygen is reduced to hydrogen peroxide, which in turn
is reduced to water, at a total expense of four equivalents of reduced glutathione. If
glucose-6-phosphate dehydrogenase deficiency patients ingest broad beans, the limited
supply of reduced glutathione causes hydrogen peroxide to accumulate. This results in
lipid peroxidation within the cell membrane and then hemolysis.
Similar redox cycles can also arise from certain drugs and drug metabolites, and the
application of such drugs must be avoided in patients with favism.
Chapter 10
Triacylglycerol metabolism
10.1 Overview
Various types of lipids occur in the human body, namely 1) triacylglycerol, 2) chol-
esterol, and 3) polar lipids, which include phospholipids, glycolipids and sphin-
golipids. This chapter will focus on triacylglycerol; cholesterol will be covered in
a separate chapter. The metabolism of polar lipids will not be covered systemati-
cally.
In contrast to polar lipids and cholesterol, which are found in the membranes
of every cell, triacylglycerol is concentrated mostly in adipose (fat) tissue; minor
amounts of triacylglycerol occur in other cell types, such as liver epithelia and
skeletal muscle fibers. Yet, overall, triacylglycerol is the most abundant lipid
species, and the only one with an important role in energy metabolism.
Triacylglycerol occurs in human metabolism in two roles, namely 1) as a food-
stuff, which accounts for a significant fraction of our caloric intake, and 2) as a
store of metabolic energy. This store can be replenished using dietary triacylglyc-
erol or through endogenous synthesis from carbohydrates or proteins.
Notes: The amount of energy stored per gram of tissue is far higher in fat than in
any other tissue, for two reasons:
1. One gram of triacylglycerol itself contains more than twice as many calories
as one gram of carbohydrates or protein. This is simply because triacylglycerol
contains much less oxygen than carbohydrates, in which oxygen contributes half
the mass but essentially no metabolic energy. Similarly, the oxygen, nitrogen and
sulfur contained in protein detract from its energy density.
147
148 10 Triacylglycerol metabolism
protein 4
carbohydrates 4
triacylglycerol 9
alcohol 7
Glyceraldehyde- P Glucose
Acetyl-CoA Pyruvate
Ketone bodies
Notes: Because of its high energy density, it makes sense that most of the excess
glucose or protein is converted to fat, while only a limited fraction is stored as
glycogen. There is, however, one limitation to the usefulness of triacylglycerol: the
conversion of carbohydrate or protein to fatty acids via acetyl-CoA is more or less
a one-way street. Carbon stored as triacylglycerol is, for the most part, confined
to the limited scope of pathways on the left of the red line in this slide. Within
this pool, the carbon may be turned into free fatty acids, which can serve as fuel
for heart and skeletal muscle, or into ketone bodies, which can supply energy to
several more organs, including the brain. However, it can no longer be turned back
into glucose; or at least not efficiently so, since the dotted “bootlegger’s pathway”
that leads from ketone bodies to pyruvate (and from there again to glucose) has
limited capacity (see slide 10.4.3).
Because of the restricted metabolic scope of triacylglycerol, we cannot rely
exclusively on fat degradation in a catabolic situation. Once glycogen is used up,
continued fasting will deplete not only stored fat but also protein, most notably in
skeletal muscle, which is necessary to maintain a sufficient supply of glucose via
gluconeogenesis.
10.1 Explain the advantages and disadvantages of triacylglycerol and glycogen as stores
of metabolic energy.
10.2 Digestion and uptake of dietary triacylglycerol 149
Notes: The fatty acids found in natural fats vary both in chain length and in
the number of double bonds. The slide shows the four most abundant species;
from the left to the right, their trivial names are palmitate, stearate, oleate,
1
and linoleate. Other chain lengths and degrees of unsaturation occur. A minor
fraction of dietary fatty acids, mostly from plants, contain less than 12 carbon
atoms; these so-called medium-chain fatty acids have some peculiar metabolic
properties that make them therapeutically useful in certain diseases (slide 10.3.9).
Notes: Detergents are amphiphilic molecules that are freely soluble in water at low
concentrations but reversibly aggregate into micelles at higher ones. When mixed
with fat, monomeric detergent molecules penetrate the fat particles and break
them up into mixed micelles; in this form, the fat becomes amenable to cleavage
by lipases. The fatty acids released by enzymatic hydrolysis act as detergents
themselves and will aid in the solubilization of remaining fat during digestion.2
1
Linolenic acid looks like linoleic acid but has an additional double bond between the third and
the fourth carbon from the far end. Such ω3-fatty acids are required but cannot be synthesized in
human metabolism; they are therefore essential.
2
Curd soap is prepared simply by alkaline hydrolysis of fat; it consists of the sodium salts of the
fatty acids released.
150 10 Triacylglycerol metabolism
Notes: After solubilization and lipase digestion, monoacylglycerol and free fatty
acids are taken up by epithelial cells in the mucous membrane of the small in-
testine. What happens to them once inside is somewhat surprising: they are
immediately converted back to triacylglycerol. This involves the transient activa-
tion of fatty acids to acyl-CoA (see slide 10.3.1) at the expense of ATP.4 The newly
formed fat is then combined with protein molecules called apolipoproteins into
lipoprotein particles, such that the proteins form a hydrophilic shell around the
lipid core. Some phospholipids are included as well and complete the hydrophilic
shell.
3
It is this property that makes them useful in treating laundry also.
4
Considering that the resynthesis immediately after cleavage is energetically costly, one might
wonder why these two steps are necessary; it might seem more economical to absorb fat molecules
without cleavage. My tentative interpretation is that the complete dispersal and degradation of
triglycerides serves as a security screen. If droplets of ingested fat were allowed to enter the system
wholesale, a lot of fat-soluble, potentially toxic compounds dissolved in them could sneak into the
system unchecked.
10.2 Digestion and uptake of dietary triacylglycerol 151
Lipoproteins occur in various subtypes (see slide 11.4.2). The specific type formed
at this stage, the chylomicrons, are the largest of all lipoproteins, with a molecular
mass of up to 1010 Dalton, a diameter up to 1 µm, and approximately 107 molecu-
les of triacylglycerol. The chylomicrons also transport dietary cholesterol; this is
discussed in slide 11.4.3.
Like glucose and other solutes taken up from the gut, the chylomicrons are re-
leased into the extracellular space at the basolateral side of the intestinal epithelia.
However, unlike those solutes, the chylomicrons are not drained toward the liver
via the portal vein, but instead are drained via the lymphatics. This is explained in
the next two slides.
10.2.4 The lymphatics drain excess fluid from the interstitial space
artery
lymph drainage
capillary
lymph
vessel
vein
Notes: This slide and the next one introduce a bit of background to explain how
chylomicrons are transported from the intestine to the systemic circulation.
The capillaries of the blood circulation are porous, and the hydrostatic pres-
sure within them drives the filtration of plasma fluid into the interstitial space.
Since the pores in the capillary walls are small, filtration is limited to water and
152 10 Triacylglycerol metabolism
small solutes. Albumin and other plasma proteins are not filtrated and therefore
maintain an osmotic pressure gradient that opposes and mostly compensates for
hydrostatic filtration. The fraction of the filtrate that is not recovered through
osmosis is drained by lymphatic vessels and then back to the venous side of the
systemic circulation.5
10.2.5 Chylomicrons are drained from the intestine through the lymphatics,
bypassing the liver
Notes: Just as plasma proteins are excluded from diffusing out of capillaries, the
chylomicrons are excluded from diffusing into them. Chylomicrons thus cannot
enter the circulation directly and must instead be drained through the lymphatic
system. The thoracic duct, which is the major effluent of the entire lymphatic
system, joins one of the major veins just a short distance upstream of the heart,
but downstream of the liver. Therefore, unlike glucose and other small molecules
that are taken up in the intestines, chylomicrons bypass the portal circulation and
the liver. They will, however, reach the liver via the systemic circulation at a later
stage (see next slide).
Notes: Once the chylomicrons have entered the circulation, the capillary wall bar-
rier must again be overcome in the delivery of triacylglycerol to extravascular cells.
This is accomplished with the help of lipoprotein lipase, which is located on the en-
dothelial surface. It binds the chylomicrons and extracts triacylglycerol from them,
which it then cleaves again to fatty acids and glycerol. These small molecules can
cross the endothelial barrier by diffusion and reach the cells in the surrounding
tissue.
5
This slow but steady flow of fluid through all tissues is also important for immune surveillance:
when a lymph vessel traverses a lymph node, the resident macrophages and lymphocytes sample
the lymph fluid for unusual antigens that would signal an infection upstream, and will promptly
mount an immune response to such antigens.
10.3 Utilization of fatty acids: β-oxidation 153
In adipose cells, the fatty acids are combined with glycerol yet again for storage. In
other cell types, most notably muscle cells, they may either be stored or degraded
directly to acetyl-CoA, which is then consumed in the TCA cycle and the respiratory
chain. The remnants of chylomicrons, depleted of most of their triacylglycerol, are
captured by the liver, endocytosed, and degraded. The cholesterol and remaining
fat released in the process is either utilized in the liver or repackaged into other
lipoprotein particles.6
10.2 Describe the processes involved in triacylglycerol digestion, absorption and storage.
As briefly mentioned before, the utilization of fatty acids occurs by way of conver-
sion to acetyl-CoA, which is accomplished in β-oxidation. This pathway runs in
the mitochondria, so the first task after cellular uptake of the fatty acid molecule
is to get it into the mitochondrion.
Notes: Fatty acids are initially activated to fatty acyl-CoA in the cytosol. This
is also the form in which they enter degradation by β-oxidation. However, dur-
ing transport, the CoA-moiety is transiently replaced by carnitine. This slide
shows the structures of both the CoA- and the carnitine-activated forms; the entire
transport process is outlined in the next one.
6
At this point, we might again wonder why the transport of fat from the intestines to other tissues
is so complicated—it might seem easier to do just pour the free fatty acids into the bloodstream. One
reason is that free fatty acids are toxic. As mentioned before, they are detergents—and detergents
dissolve cell membranes. (Interestingly, the membranes of the intestinal cells withstand very high
concentrations of bile and fatty acids! They manage this due to their high content of ceramide,
which is also found in the keratin of the skin.) However, as we will see later, fatty acids with shorter
acyl chains are indeed transported in free form.
154 10 Triacylglycerol metabolism
Acyl-CoA
O O O
O N N S
H H
OH
−O P O
NH2
O
O
−O P O N
N
−O
O
O N N
O
O
N+
OH OH
Acyl-carnitine
Notes: Fatty acids are activated in the cytosol to acyl-CoA by acyl thiokinase, also
known as acyl-CoA synthetase. After transport across the outer mitochondrial
membrane, the acyl group is transferred to carnitine by carnitine acyltransferase.
Exchange for free carnitine transports the acylcarnitine molecule into the mito-
chondrial matrix, where carnitine is replaced again with coenzyme A by a second
acyl transferase.
AMP + PPi
Fatty acid
Acyl-carnitine Acyl-carnitine
One unusual aspect of this transport process is that the energy of the thioester
bond in acyl-CoA seems to be sufficiently well preserved in the ester bond of acyl-
carnitine to allow the exchange reaction to be reversed inside the mitochondrion.
Carboxyl esters bonds don’t usually have a sufficiently high energy content for
that. I once stumbled upon a theoretical paper explaining why carnitine is special
in this regard, but it went straight over my head, and I therefore cannot give you
an explanation.
10.3 Utilization of fatty acids: β-oxidation 155
O β
CoA
S
FAD
1
FADH2 O
CoA
S
H2 O
2
O OH
CoA
S
NAD+
3
NADH+H+ O O
CoA
S
CoA−SH
4
O O β′
CoA CoA
S S
Notes: The term β-oxidation refers to the Greek lettering of the carbons in organic
molecules: the α carbon is right beside a functional group, and the β carbon is the
next one. It is at the second carbon from the thioester, then, where the action is in
β-oxidation. The reactions are as follows:
1. The fatty acyl-CoA molecule is first dehydrogenated between the α and the
β carbon atoms by acyl-CoA dehydrogenase. FAD accepts the hydrogen
abstracted and is reduced to FADH2 . This yields 2-trans-enoyl-CoA.
2. The trans double bond just created is hydrated—that is, water is added to
it—by enoyl-CoA hydratase, which yields hydroxyacyl-CoA. The α carbon is
now once more fully reduced.
3. The β-hydroxyl group is converted to a keto group by hydroxyacyl-CoA dehy-
drogenase. NAD+ accepts the hydrogen. The product is β-ketoacyl-CoA.
4. Thiolase introduces a new molecule of coenzyme A to cleave the β-ketoacyl-
CoA, which releases acetyl-CoA and a new, shortened acyl-CoA that enters the
next cycle of β-oxidation.
The process is repeated until the fatty acid is completely broken down. In the
case of acyl chains with even numbers of carbons, this will yield acetyl-CoA only,
whereas those with odd numbers of carbons will yield one molecule of propionyl-
CoA in the final thiolase step. There is a special pathway to take care of the
propionyl-CoA, which is surprisingly complicated (see slide 10.3.6).
10.3 Draw the reactions in β-oxidation.
156 10 Triacylglycerol metabolism
Notes: You may have noticed the similarities of the enzyme reactions discussed
above to some others we have seen before. In the first three steps, the similarities
to reactions from the citric acid cycle are quite straightforward. Also notice the
consistent use of redox coenzymes (NAD+ and FAD) in both pathways: Wherever
a CH−OH bond is dehydrogenated, NAD+ is employed as the cosubstrate,7 while
FAD is used where dehydrogenation occurs across a CH−CH bond.
BH +
Enzyme
S− O
CoA
S
H+
B
O
Enzyme
S
−S CoA
O
CoA
S
Notes: The thiolase mechanism does not have a closely analogous precedent
among the reactions we have seen so far. However, if we look at the individual
steps of the thiolase reaction, we can still recognize some familiar features:
7
This also holds in other pathways and for CH−NH bonds. The only exception I’m aware of is
the dehydrogenation of glycerophosphate in the glycerophosphate shuttle.
10.3 Utilization of fatty acids: β-oxidation 157
Propionyl-CoA S-methylmalonyl-CoA
ATP ADP
O O O
CoA CoA
S 1 S OH
CO2
O O O
3
CoA OH CoA
S S OH
O
Succinyl-CoA R-methylmalonyl-CoA
Notes: Fatty acids with odd numbers of carbon atoms yield one molecule of propi-
onyl-CoA as the final degradation product. This metabolite has a rather elaborate
degradative pathway:
1. Initially, propionyl-CoA is converted to S-methylmalonyl-CoA by propionyl-
CoA carboxylase. This reaction uses CO2 and ATP, with biotin serving as a
coenzyme; the mechanism resembles that of pyruvate carboxylase (slide 7.2.4).
2. S-methylmalonyl-CoA is converted to its enantiomer R-methylmalonyl-CoA
by methylmalonyl-CoA racemase.
3. Finally, one carboxyl group is transplanted within the molecule by methyl-
malonyl-CoA mutase to yield succinyl-CoA. This is a frighteningly complex re-
action; we will skip the gory details and just note that it requires vitamin B12 ,
in the form of adenosylcobalamin, as a coenzyme. When this vitamin is lacking,
methylmalonyl-CoA backs up and is hydrolyzed to free methylmalonate, which
may inhibit gluconeogenesis (see slide 7.4.1).
Note that succinyl-CoA is a citric acid cycle intermediate. It can therefore enter
gluconeogenesis (see slide 7.1), so that propionyl-CoA is an exception to the rule
that carbon from fatty acids cannot be used for gluconeogenesis. However, since
fatty acids with odd numbers of carbons amount to a small fraction of all fatty
acids, this exception is not quantitatively very important.
158 10 Triacylglycerol metabolism
10.4 If we assume that oxidation of NADH yields 3 equivalents of ATP and oxidation
of FADH2 yields 2, how many equivalents of ATP does the complete oxidation of one
molecule of palmitoyl-CoA provide?
triacylglycerol
acetyl-CoA
Notes: When triacylglycerol is mobilized from the fat tissue, its hydrolysis to free
fatty acids and glycerol is initiated by an enzyme named hormone-sensitive lipase.
This lipase is stimulated downstream of glucagon and epinephrine, the antagonist
hormones of insulin. As see before in carbohydrate metabolism (slides 7.5.4 and
8.4.2), the two hormones activate adenylate cyclase and protein kinase A. The
fatty acids produced by the lipase are released into the bloodstream, where they
bind to albumin for transport. The fatty acids can be utilized in two different
ways:
1. Directly, that is, they are taken up by the energy-requiring target tissue
and degraded by β-oxidation. We noted above that β-oxidation is preceded by
the translocation to the mitochondrion, which requires carnitine. About 95% of
all carnitine is found in skeletal muscle,8 which implies that muscle tissue is the
major client for direct utilization. The heart muscle cells also consume fatty acids
through β-oxidation.
2. Indirectly, after initial conversion to ketone bodies in the liver. This term
comprises two small organic acids, acetoacetate and β-hydroxybutyrate, as well
as acetone. The brain, which in the well-fed state only utilizes glucose for ATP
regeneration, is able to replace up to 50% of its usual glucose consumption with
ketone bodies during starvation. The brain is responsible for approximately 20%
of our total energy consumption at rest, and therefore is a major consumer of
ketone bodies. Other major clients are the heart and skeletal muscle.
8
The word carnis is Latin for meat—as in chili con carne.
10.3 Utilization of fatty acids: β-oxidation 159
The second cleavage product of fat, glycerol, is released into the bloodstream
and picked up by the liver as well. It is phosphorylated by glycerol kinase to
glycerophosphate. As we have seen before (slide 6.9.3), this metabolite can be
dehydrogenated to dihydroxyacetone phosphate, which may enter gluconeogene-
sis. Glycerol released from fat constitutes a minor source of glucose in times of
starvation.
The pattern of fat usage sketched out here holds for the bulk of all adipose
tissue, which is white. An exception from this pattern is brown fat tissue, which is
covered in the next slide.
Notes: Cells in white fat tissue owe their color to the fact that they contain little
else than large, single triacylglycerol droplets. Intensely colored tissues, other than
the pigment cells of the skin, are rich in heme or cytochromes. This is also the case
with brown fat tissue. In addition to multiple small lipid droplets (Ld), they contain
an abundance of mitochondria (M), which in turn are rich in cytochromes. The
mitochondria in this particular tissue also contain a high amount of an uncoupling
protein (slide 6.3.1) called thermogenin.
In contrast to white fat cells, brown fat cells do not release fatty acids into
the circulation but instead carry out β-oxidation themselves. The accumulated
hydrogen is oxidized in the respiratory chain. The resulting proton-motive force is
not used for ATP synthesis, but is instead simply dissipated as heat—the purpose
of brown fat is heat production.
There is little brown fat in adult humans or most other adult mammals. How-
ever, there is a substantial amount in newborns, who because of their higher
surface-to-volume ratio, and their helplessness, are at greater risk of hypother-
mia. Brown fat is also found in hibernating animal species such as groundhog,
which need it to reheat themselves to operating temperature during arousal from
hibernation.
160 10 Triacylglycerol metabolism
FAD FADH2
acetyl-CoA
fatty acids acetyl-CoA
succinate
+ + acetoacetyl-CoA
NAD NADH+H citrate
β-hydroxy-
acetoacetate
butyrate succinyl-CoA
acetoacetate
β-hydroxy-butyrate
Notes: The key idea of ketone body metabolism is to convert free fatty acids into
more water-soluble substrates that are easier to transport and to metabolize. This
scheme gives an overview of organ relationships and pathways; note that the
reactions are not stoichiometrically balanced.
10.4 Ketone body metabolism 161
Ketone bodies are formed mostly in the liver. As stated earlier, the first
stage of ketogenesis consists in β-oxidation. The resulting acetyl-CoA is turned
into acetoacetate along the pathway detailed in the next slide. The subsequent
reduction of acetoacetate to β-hydroxybutyrate allows the liver to dispose of some
of the surplus hydrogen that accumulates during β-oxidation. Both acetoacetate
and β-hydroxybutyrate are released into the circulation.
Utilization of ketone bodies in the brain, muscle and other tissues is quite
straightforward. β-Hydroxybutyrate is dehydrogenated again to acetoacetate,
which steals coenzyme A from succinyl-CoA to become acetoacetyl-CoA. Thio-
lase then cleaves acetoacetyl-CoA to two molecules of acetyl-CoA, which enter the
TCA cycle.
2 acetyl-CoA acetoacetyl-CoA
O O O O
1
CoA CoA CoA
S S S
CoA-SH
S
CoA
H2 O
2 O
CoA-SH
+ +
NAD NADH+H
O OH
HO O 3
CoA
4 S
OH OH OH
O
O O CoA O
S
β-hydroxybutyrate acetoacetate HMG-CoA
Notes: This slide details the mitochondrial pathway that converts acetyl-CoA to
ketone bodies. It involves the following steps:
1. formation of acetoacetyl-CoA from two molecules of acetyl-CoA by thiolase.
This step is the reversal of the final step in β-oxidation;
2. formation of hydroxymethylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase;
3. release of acetoacetate by HMG-CoA lyase; and
4. reduction of acetoacetate to β-hydroxybutyrate by β-hydroxybutyrate dehy-
drogenase.
serum is greater when ketogenesis has been induced, suggesting the existence of a
specific enzyme activity. A bacterial acetoacetate decarboxylase is known and has
been studied in molecular detail; however, the hypothetical mammalian enzyme
remains incompletely characterized [47].
80
40
CO2
serum ultrafiltrate
20
O
Acetone
0
0 20 40 60 80 100 120
Incubation time (min)
Notes: While many textbooks, even ones of recent vintage, continue to spread the
myth of acetone being a useless metabolic dead end, it has been known for some
time that acetone can be converted to pyruvate, which can then enter gluconeoge-
nesis [48]. Thus, acetone provides a back door for fatty acyl carbon to be turned
back into glucose. The capacity of this pathway appears to be limited; it has been
estimated that up to 11% of endogenously produced glucose may be derived from
acetone in fasting humans [49].
C O C O HO CH
NAD+
NADH
OH OH
C O HO CH HO CH
The first step of the pathway shown here is catalyzed by the enzyme cytochrome
P450 type 2E1. This enzyme can also metabolize ethanol and is transcriptionally
induced by both acetone and ethanol. The enzyme that converts acetol to propane-
diol has not been characterized; the subsequent steps are catalyzed by alcohol
10.4 Ketone body metabolism 163
N
N
N 40
N
1 10 100
Pentylenetetrazole (PTZ) Metabolite injected (mmol/kg)
9
A second pathway from acetol to pyruvate involves the oxidation of acetol by cytochrome
P450 2E1 to methylglyoxal, which is then converted to d-lactate through addition and subsequent
hydrolysis of glutathione [48].
10
Considering the very high concentrations of ketone bodies in diabetic coma, one might assume
that acetone contributes to the causation of unconsciousness in this condition. However, a clinical
study on this question has concluded that acetone is likely not a dominant factor [50].
164 10 Triacylglycerol metabolism
Fatty acids can be synthesized from acetyl-CoA. This is the major pathway for
utilizing excess dietary carbohydrates and protein. Fatty acid synthesis occurs
mainly in the fat tissue and the liver. It runs in the cytosol, which keeps it apart
from mitochondrial β-oxidation. In broad outline, fatty acid synthesis is a reversal
of β-oxidation: two carbon atoms at a time are added to a growing fatty acid
molecule, and the new β carbon is then reduced to the alkane level. However, the
mechanistic details are somewhat different.
The bulk of the work in fatty acid synthesis is accomplished by a single en-
zyme, fatty acid synthase, which is quite an amazing molecule: it combines six
active sites with eight distinct catalytic activities on a single polypeptide chain. Its
product is palmitic acid (hexadecanoic acid). As stated before, fatty acids vary in
their chain lengths and degree of bond saturation. These variants are derived from
palmitate through chain elongation and desaturation, which are accomplished by
separate enzymes called elongases and desaturases.
O ATP ADP+Pi O O
CoA CoA
S S OH
CO2
Acetyl-CoA Malonyl-CoA
Notes: The only reaction in palmitate synthesis that is not carried out by fatty acid
synthase itself is catalyzed by acetyl-CoA carboxylase. I hope that by now you rec-
ognize the pattern: CO2 is fixed, and ATP is expended—another biotin-dependent
reaction, working in the same way as pyruvate carboxylase and propionyl-CoA
carboxylase.
The acetyl-CoA carboxylase reaction is performed for each C2 subunit that
is added to the growing fatty acid, except the very first one. As with pyruvate
carboxylase in gluconeogenesis, the attachment of CO2 is transient, and one can
think of it simply as an activation step that facilitates subsequent C−C bond
formation.
Notes: All further reactions are catalyzed by fatty acid synthase. In animals, this
is a large molecule with multiple active sites located on a single polypeptide chain.
Two of these polypeptides form an active dimer. The active sites are highlighted
in the structure (rendered from 2vz8.pdb), and their locations along the sequence
of the fatty acid synthase molecule are given as acronyms, as follows: KS, ketoacyl
synthase; MAT, malonyl-acetyltransferase; KR, ketoacyl reductase; DH, hydroxyacyl
dehydratase; ER, enoylreductase. The numbers indicate the place of each active
site in the sequence of reactions. ACP is acyl carrier protein, and TE thioesterase;
10.5 Fatty acid synthesis 165
these two elements were not resolved in the crystal structure of the protein, and
their locations are approximate.
Notes: Throughout the repetitive cycle of reactions involved in the synthesis, the
growing fatty acyl chain remains covalently attached to the enzyme. Attachment
is made through a phosphopantetheine group, which functions as a flexible tether,
allowing the acyl chain to travel and visit the various active sites on the enzyme
in turn. This is reminiscent of the lipoamide moiety in pyruvate dehydrogenase
and of biotin-dependent carboxylases.11 The phosphopantetheine tether group is
actually not new to us, since it also occurs in coenzyme A (see slide 10.3.1).
O O O OH
O
ACP O P O N N S
H H
O− OH
Notes: The reactions catalyzed by fatty acid synthase are shown in this slide and
the next one.
1. Malonyl-acetyltransferase loads the first acetyl-group onto the ACP moiety. In
both the substrate and the product, the acetyl group is bound as a thioester,
which makes this an easy reaction.
11
In E. coli, the phosphopantetheine group is associated with a separate small protein, the acyl
carrier protein (ACP). In mammalian fatty acyl synthesis, ACP is not a separate protein but is part
of the synthase molecule itself.
166 10 Triacylglycerol metabolism
Notes: The synthesis continues from the last slide, with the following reactions:
6. hydroxyacyl dehydratase eliminates water, and
10.5 Fatty acid synthesis 167
7. enoylreductase reduces the ensuing double bond. These two steps yield
butyrate.
8. Ketoacyl synthase acquires the butyrate, freeing up ACP.
9. Malonyl-acetyltransferase acquires the next malonyl residue from malonyl-
CoA, which is again supplied by acetyl-CoA carboxylase.
From this point on, the the cycle repeats—ketoacyl synthase decarboxylates
malonate and forms the next β-keto acid, ketoacyl reductase reduces it, and so on.
The cycle executes seven times overall, which results in the formation of palmitate;
the palmitoyl residue is then released as free palmitate by the thioesterase activity
of fatty acid synthase.
10.7 Give an outline of fatty acid synthesis.
mitochondria cytosol
citrate citrate
oxaloacetate oxaloacetate
NADH+H+ NADH+H+
NAD+ NAD+
malate malate
Notes: We have noted above that fatty acid synthesis occurs in the cytosol. Since
acetyl-CoA is formed in the mitochondrion by pyruvate dehydrogenase, it needs
to get out of the mitochondrion and into the cytosol.
We saw before that there is a similar problem in β-oxidation. In that case,
the transport of acyl-CoA to the mitochondria is accomplished by the carnitine
carrier system (slide 10.3.2). Since all reactions in the latter system are reversible,
and acetyl-CoA is also an acyl-CoA, we might expect that the carnitine carrier
system could help us out here as well. On the other hand, if transport of acyl-CoA
molecules went both ways, this might facilitate a futile cycle of fatty acid synthesis
and degradation.
It turns out that acetyl-CoA is indeed transported by other means. One of them
is illustrated in this slide. Here, citrate is exported by the tricarboxylate carrier sys-
tem. Cleavage by ATP-citrate lyase reverts the citrate synthase reaction, producing
acetyl-CoA and oxaloacetate. The latter is reduced by malate dehydrogenase, and
malate is exchanged for citrate. As you can see, this process will also transport one
NADH equivalent to the mitochondria, which is usually accomplished by dedicated
shuttle systems (section 6.9).
168 10 Triacylglycerol metabolism
CoA-SH
acetyl-CoA
CoA-SH CoA-SH
HMG-CoA acetoacetyl-CoA
AMP+PPi
acetoacetate acetoacetate
mitochondria cytosol
duce double bonds closer than 9 carbons away from the ω end—that is, non-car-
boxyl end—of the substrate. Since we require ω-3 and ω-6 fatty acids as synthetic
precursors of membrane lipids and of prostaglandins, such fatty acids are es-
sential, that is, they must be obtained from the diet. All ω-3 and ω-6 fatty
acids ultimately originate in plant metabolism, even if we may ingest them with
animal-derived food.
β-keto acid OH
O O
H⊕
⊖S Cys FAS
O
NH2
cerulenin
O O
Cys FAS
HO S
NH2
covalent adduct
O O
Notes: Cerulenin is a fungal antibiotic that binds and irreversibly inactivates fatty
acid synthase. Its structure resembles the β-ketoacyl intermediates of fatty acid
synthesis, and indeed cerulenin binds to the ketoacyl synthetase site of the enzyme.
The cysteine residue in this site then reacts with the epoxide ring in cerulenin and
is alkylated.
While epoxide derivatives are not terribly popular as drugs because they tend
to be quite reactive and often toxic, there is presently some interest in the develop-
ment of fatty acid synthase inhibitors, modeled on the structure of cerulenin, for
the treatment of obesity. While this does not strike me as a particularly promis-
ing idea,12 a more interesting application is in the treatment of cancer. This is
discussed in the next slide.
10.5.10 Fatty acid synthase inhibition slows tumor growth in mouse experi-
ments
Notes: Most non-cancerous cells do not express fatty acid synthase, relying instead
on fatty acids supplied by the liver and fat tissue. However, fatty acid synthase
expression has been observed in cancer cells. The cells probably use these fatty
12
I look forward to the discoveries of metabolic derailments these drugs will cause in the face of
continued excess caloric intake. What is going to become of the surplus acetyl-CoA when fatty acid
synthesis is inhibited: Ketone bodies? Cholesterol? Will glycolysis be backed up and diabetes be
induced? All of the above?
170 10 Triacylglycerol metabolism
acids to supply their synthesis of membrane glyco- and phospholipids, which they
require for proliferation. In any event, inhibition of fatty acid synthase in tumor
cells can drive them into apoptosis (programmed cell death).
OH
0.3
O O
0.2
O
0.1 compound 7
OH
O
0
OH 0 10 20 30 40
compound 7 OH days of treatment
This slide shows an experimental synthetic drug (with the provisional name “com-
pound 7”) that is somewhat similar to polyphenols occurring in green tea and
other natural sources. In mice experiments, it substantially reduces the growth
rate of transplanted cancer. While such experimental cancers are notorious for
being easier to treat than actual clinical cancer in humans, the results suggest that
this line of research is worth pursuing. Figure prepared from original data in [57].
Glucose
Oxaloacetate Citrate
Malate Isocitrate
Fumarate α-Ketoglutarate
Glyoxylate
Succinate Succinyl-CoA
Acetyl-CoA
Notes: It was discussed before that carbon contained in fatty acids cannot be uti-
lized efficiently for gluconeogenesis, since there is no efficient pathway to convert
the acetyl-CoA that results from their breakdown into TCA cycle intermediates
(see section 5.6 and slide 10.1.2). Interestingly, however, plants do have a straight-
forward pathway to do just this, namely, the glyoxylate cycle, which is an ancillary
road to the TCA cycle.
In the glyoxylate cycle, the two decarboxylation steps of the TCA cycle are
skipped, and an entry point for a second molecule of acetyl-CoA is created. In this
10.7 Answers to practice questions 171
manner, plants are able to use two molecules of acetyl-CoA for the net synthesis
of one C4 TCA cycle intermediate.
H3 C S CoA H2 O H2 C OH
HO C COO−
H
O C COO− CoA-SH
H
Notes: The cycle involves two reactions, both of which are mechanistically similar
to citrate synthase:
1. Isocitrate is split into succinate and glyoxylate by isocitrate lyase. Since the
isocitrate dehydrogenase and the α-ketoglutarate dehydrogenase reactions
are bypassed, the loss of two carbons as CO2 is avoided; these carbons are
retained in the form of glyoxylate.
2. Glyoxylate combines with the second acetyl-CoA molecule to form malate.
This reaction is catalyzed by malate synthase, and like the citrate synthase
reaction it is pushed forward by the concomitant hydrolysis of coenzyme A.
You know that many plant seeds are very rich in oil—that is, fat. The glyoxylate
cycle enables plant seeds to store metabolic energy and carbon at high density
as fat, and to use it for the synthesis of glucose and other carbohydrates during
germination.
Cholesterol metabolism
173
174 11 Cholesterol metabolism
The pathway of cholesterol synthesis is quite elaborate. We will start with a general
outline and then go through most, but not all of the reactions in detail.
HMG-CoA
reductase
acetyl-CoA HMG-CoA mevalonate
2NADPH + 2H+
3
H2 O + CoA-SH 2NADP+
⊖
P
P O O 3ATP O OH
5 ⊖O
4 ⊖O
O P P O P P OH
CO2 3ADP
isopentenyl- mevalonate
pyrophosphate
Notes: The reactions shown in this slide are catalyzed by thiolase (1), HMG-CoA
synthase (2), HMG-CoA reductase (3), mevalonate kinase and phosphomevalonate
kinase (4), and diphosphomevalonate decarboxylase (5). In the subsequent
steps of the pathway, six molecules of isopentenyl-pyrophosphate are used for the
synthesis of one cholesterol molecule.
Notes: The next stage begins with the conversion of one molecule of isopentenyl-
pyrophosphate to dimethylallyl-pyrophosphate, catalyzed by the isopentenyl-pyro-
phosphate isomerase. The product is condensed with another molecule of isopen-
tenyl-pyrophosphate to yield geranyl-pyrophosphate. In this reaction, catalyzed by
geranyl-pyrophosphate synthase, the pyrophosphate of the first substrate serves
as a leaving group. The resulting carbocation reacts with the double bond of the
second substrate.
176 11 Cholesterol metabolism
O P P isopentenyl-pyrophosphate
O P P ⊕ O P P
dimethylallyl-pyrophosphate PPi
⊕
O P P
H⊕
geranyl-pyrophosphate O P P
geranyl-pyrophosphate isopentenyl-pyrophosphate
O P P O P P
PPi H⊕
O P P farnesyl-pyrophosphate
P P O
NADPH + H+
NADP+ 2 PPi
squalene
11.2 Cholesterol synthesis 177
squalene
NADPH+H+ NADP+
O
⊕H squalene epoxide
O2 H2O
⊕
lanosterol
HO H+ HO
Notes: The reactions shown here are catalyzed by squalene epoxidase and lanos-
terol synthase. The rearrangement indicated by the dashed arrow is not a real
reaction—we just rotate a couple of single bonds to show how the pieces fall into
place for the subsequent cyclization.
The oxygen is introduced by squalene epoxidase, a cytochrome P450 enzyme.
Such enzymes use NADPH to reduce one of the two atoms of molecular oxygen,
while retaining the other one in a highly reactive state, which they then use toward
their specific purposes (see slide 18.2). Squalene synthase inserts its active oxygen
into a C−−C double bond of the substrate to form an epoxide. The subsequent
cleavage of the epoxide by lanosterol synthase starts a cascade of reactions that
goes from one end of the molecule to the other, closing all four rings of the sterol
skeleton in the process. Note that a methyl group also changes its place on the
sterol ring; the reaction mechanism is quite intricate.
drug triparanol, which was once used to treat hypercholesterolemia before being
withdrawn due to toxicity.
HO HO HO
11.3 What is the role of cytochrome P450 enzymes in the synthesis of cholesterol?
hν 25-OH 1-OH
HO
HO HO OH
remarkably pale skin of people of British or Irish descent tells you all you need to
know about the weather in those places. Avoid.
11.4 What are the metabolic roles of 7-dehydrocholesterol?
Notes: The sterol response element (SRE) is a DNA consensus sequence that con-
trols the transcription of HMG-CoA reductase. The corresponding SRE-binding
protein (SREBP) is initially embedded in the ER membrane, and thus evidently
unable to get in touch with its DNA target. SREBP is bound to a second protein,
namely, SREBP cleavage activating protein (SCAP). This protein is the actual chol-
esterol sensor; it can adopt two different conformations, depending on the content
of cholesterol in the surrounding membrane. The conformation that predominates
at high cholesterol content lets SCAP bind to a third protein, INSIG.1 When this
ternary complex forms, it is rapidly targeted toward proteolytic degradation, and
that is the end of it.
At low cholesterol concentrations, however, SCAP does not bind to INSIG, and
this is when things get interesting, as shown in the next slide.
1
INSIG stands for “insulin-induced gene.”
11.3 Regulation of cholesterol synthesis 181
Notes: Once SREBP reaches the Golgi, it is ambushed and cleaved by two specific
proteases (S1P and S2P). Cleavage releases the DNA-binding domain of the protein
from the membrane. This fragment then moves across the cytosol and enters the
nucleus, where it binds to SRE sequence elements that increase the expression
of HMG-CoA reductase and also of various other enzymes from the cholesterol
synthesis pathway [58].
Another protein that is upregulated by SREBP and SRE is the LDL receptor, a
membrane protein that mediates endocytosis of low density lipoprotein (LDL;
see slide 11.4.7). In cells that do not synthesize cholesterol themselves, SREBP
upregulates transcription of the LDL receptor,
182 11 Cholesterol metabolism
Like other lipids, cholesterol has low water solubility and therefore requires special
mechanisms and vehicles for transport. In the bloodstream, both cholesterol and
triacylglycerol are transported within lipoproteins.
apolipoproteins
phospholipids, triacylglycerol,
free cholesterol cholesterol esters
Notes: We had already encountered one type of lipoprotein, namely, the chylomi-
crons, which are formed in the intestinal mucosa (see slide 10.2.3). Different
lipoprotein types vary in composition and particle size, but the overall structure is
similar for all of them. The surface consists mainly of phospholipids, which form
a monolayer.
Apolipoproteins are embedded into the surface of the particle. These protein
molecules mainly serve as “address tags” that mediate the interaction with target
molecules and cells, such as lipoprotein lipase on endothelial cells (slide 10.2.6) or
the LDL receptor (see later). Some apolipoproteins are stably associated with their
lipoprotein particles; for example, a given molecule of apolipoprotein B remains
associated with the same chylomicron or VLDL (very low density lipoprotein, see
below) particle throughout its lifetime. Exchangeable apolipoproteins have regula-
tory roles; for example, apolipoprotein CII reversibly associates with VLDL particles
and activates their interaction with lipoprotein lipase.
11.4 Cholesterol transport 183
Notes: As you will notice, the variation in density between the various lipoprote-
ins is modest, but it is large enough to allow their separation by density gradient
centrifugation, and they are classified according to their behavior in this fraction-
ation procedure. As noted before, ‘LDL’ stands for low density lipoprotein; ‘HDL’
means high density lipoprotein, and ‘VLDL’ is very low density lipoprotein. The
differences in density arise from two circumstances:
1. Lipids are lighter than protein. Within the particles, proteins are found only
at the surface, whereas the interior contains only lipids; therefore, the frac-
tional content of protein is lower with larger particles than with smaller ones.
Chylomicrons are the largest and least dense lipoprotein species.
2. Triacylglycerol is lighter, but cholesterol is heavier than water. Accordingly, a
high content of cholesterol in the lipid fraction will also increase the overall
density.
Note the central role of the liver, which orchestrates most of the lipid transport,
with the exception of intestinal lipid uptake and packaging into chylomicrons. We
will now first look at the uptake of cholesterol in the intestine and then at its
transport to other organs.
11.4.3 Two membrane proteins control the uptake of sterols from the intes-
tine
Notes: The uptake of cholesterol by intestinal epithelial cells begins with endo-
cytosis. This process is controlled by NPC1L1, a membrane protein which is
deficient in a lipid storage disorder known as Niemann-Pick disease. NPC1L1 is a
sterol sensor and promotes cholesterol uptake through endocytosis.
From the endocytotic vesicles, cholesterol is transferred to the endoplasmic
reticulum by the microsomal triglyceride transfer protein (MTTP). Acylation by acyl-
CoA cholesterol acyltransferase (ACAT) yields a cholesterol ester, which is loaded
into a nascent chylomicron together with triacylglycerol. After the chylomicrons
184 11 Cholesterol metabolism
have been released from the intestinal cells and reached the circulation via the
lymphatics, most of their triacylglycerol is depleted by capillary lipoprotein lipase.
The cholesterol stays behind in the chylomicron remnants, which are taken up and
utilized in the liver (see slide 10.2.6).
In contrast to what its name suggests, MTTP transports not only triacylglycerol
but also sterols. Mutational inactivation of this protein results in abetalipopro-
teinemia.2 Such patients have reduced levels of chylomicrons and are affected by
malabsorption of lipids and of lipid-soluble vitamins.
The NPC1L1-mediated uptake of cholesterol by endocytosis does not discrimi-
nate between cholesterol and other, structurally similar sterols derived from plants.
After uptake, the latter are diverted toward a transport protein in the apical mem-
brane, ABCA5/8, which expels them right back into the gut lumen through active
transport.
11.6 How is cholesterol taken up in the small intestine?
Notes: Plants contain little cholesterol but instead contain a variety of structurally
similar sterols. The sterol ring is the same as with cholesterol in all sterols shown,
but the tails are somewhat different.
Plant sterols compete with cholesterol for “space” inside the cytoplasmic mem-
brane of intestinal cells, and therefore reduce the rate of cholesterol absorption by
endocytosis. Dietary application of sitosterol or other plant sterols is a moderately
effective strategy to reduce cholesterol absorption.
11.7 How do plant sterols influence intestinal cholesterol uptake?
2
The name of “abetalipoproteinemia” denotes the lack of apolipoprotein B, which is the major
protein constituent of both chylomicrons and VLDL.
11.4 Cholesterol transport 185
cholesterol
ring
avenasterol
ring
tail
brassicasterol
ring
HO campesterol
ring
stigmasterol
ring
Notes: Both ABCA5/8 (previous slide) and ABCA1 (slide 11.4.7) are members of
the ATP-binding cassette or ABC family of transporters. These have a common
structural organization. Several ABC transporters have been crystallized in the
inward- and outward open conformations, and the structures provide a glimpse of
how they work.
ATP
ABC transporters often have rather broad substrate specificity and mediate the
membrane translocation of many metabolites and xenobiotics. In addition to
cholesterol and other membrane lipids, important examples are bile acids (slide
11.5.3), conjugated bilirubin (slide 17.4), drugs, and drug metabolites (18.1.3).
186 11 Cholesterol metabolism
Cancer cells often overexpress ABC transporters, which renders them resistant to
multiple anticancer drugs.
Notes: One feature that is shared by many ABC transporter substrates is their
amphiphilic nature. Most ABC transporters expel their substrates from the cytosol
to the extracellular space. In this case, the substrate initially resides within the
inner leaflet of the cytoplasmic membrane. Once it enters the inward-open confor-
mation of the transporter, the latter undergoes a transition to the outward-open
conformation, which is powered by the hydrolysis of ATP. The substrate then
leaves the transporter and diffuses into the outer membrane leaflet, from where it
may distribute to other extracellular reservoirs.
chylomicrons
LPL
LPL
chylomicron remnants VLDL LDL
LDL receptor
cholesterol
HDL
LCAT ABCA1
cholesterol
nascent HDL
Notes: The liver synthesizes cholesterol from acetyl-CoA (section 11.2). The chol-
esterol pool in liver cells also receives the dietary cholesterol, which is contained in
11.4 Cholesterol transport 187
the chylomicron remnants that are formed through the extraction of triacylglycerol
from chylomicrons by lipoprotein lipase (LPL; slide 10.2.6).
Liver cells package esterified cholesterol, together with triacylglycerol, into
particles of very low density lipoprotein (VLDL). Like chylomicrons, VLDL interacts
with lipoprotein lipase and thereby turns into intermediate (IDL) and then low
density lipoprotein (LDL).
LDL is taken up by cells in the periphery through endocytosis, which is me-
diated by the LDL receptor.3 Excess cholesterol is exported from the cell by an
active transporter (ABCA1) and delivered to high density lipoprotein (HDL), which
then carries it back to the liver. Cholesterol transport by HDL is facilitated by
lecithin-cholesterol acyltransferase (see next slide).
N⊕ N⊕
O O
O O
P P
O O⊖ O O⊖ O
OH O
O HO
O
O O
O O
LCAT
lecithin lysolecithin
Notes: The HDL particle contains the enzyme lecithin cholesterol acyltransferase,
or LCAT for short, which converts cholesterol to cholesterol esters.
The LCAT reaction occurs at the surface of HDL particles. The transfer of one
acyl chain from a lecithin (phosphatidylcholine) molecule to cholesterol produces
a cholesterol ester and lysolecithin. The significance for cholesterol transport is
illustrated in the next slide.
Cholesterol also undergoes esterification as it is packaged into chylomicrons
and VLDL inside intestinal and liver cells, respectively. In these cases, acyl-CoA
serves as the donor of the acyl residue (see slide 11.4.3).
3
LDL manages to leave the circulation by transcytosis, that is, endocytosis by the vascular en-
dothelial cells on the luminal side, followed by exocytosis on the opposite side.
188 11 Cholesterol metabolism
Bile acids are the quantitatively most important derivatives of cholesterol. Aside
from their essential role in fat digestion, they are also required to keep hydropho-
bic constituents of the bile in solution, such as unconjugated bilirubin (see section
17.4) and cholesterol itself.
cholate taurocholate O
S O
−
H O
O− N
O O
OH OH
HO OH HO OH
Notes: Cholic acid and some other bile acids are synthesized from cholesterol in
the liver. Taurocholate is produced through conjugation of cholate with taurine;
similarly, glycocholate is produced through conjugation with glycine.
11.5 Bile acid metabolism and transport 189
11.9 What kinds of molecules are derived from cholesterol in human metabolism? Which
derivatives are quantitatively the most important ones?
Notes: In an enterohepatic cycle, a substance is secreted by the liver into the bile,
passes into the intestine and is taken up again into the blood, either by passive
diffusion across cell membranes or by active transport. Since blood drained from
the intestines feeds into the portal vein, the substance will return to the liver,
where it may be captured by liver cells and once again secreted into the bile.
Bile acids are taken up by active transport in the terminal ileum, that is, in the
lowermost section of the small intestine. The efficiency of reuptake is normally
> 90 %. Only the fraction that is not recovered needs to be replaced by de novo
synthesis from cholesterol.
During their repeated passages through the intestine, some bile acids undergo
modification by microbial enzymes; an example is the formation of deoxycholate
from cholate. Such modified molecules become part of the circulating bile acid
pool.
Notes: A variety of transport proteins enable the bile acid enterohepatic cycle. Se-
cretion from the liver cell into the bile is driven by ABCC2, another ABC type trans-
porter (compare slide 11.4.5). Reuptake from the lumen of the gut is mediated by
the apical sodium-coupled bile acid transporter (ASBT). A similar transporter, the
Na+ -dependent taurocholate cotransporting polypeptide (NTCP), mediates uptake
from the blood back into the liver cell. At the basolateral membranes of both
intestinal and liver cells, organic anion transport proteins (OATPs), which have a
fairly low degree of substrate specificity, participate in bile acid transport.
190 11 Cholesterol metabolism
terolemia and that additional factors, of which there are many, should
whenever possible be considered in the context of how they relate to the
processes initiated by hypercholesterolemia [60].
Notes: The earliest readily visible atherosclerotic lesion is the fatty streak. It forms
within the wall of an artery, in the thin layer of connective tissue that is located
underneath the endothelium (the innermost cell layer of any blood vessel) and
atop the thick layer of smooth muscle that maintains the wall tension and blood
pressure.
Fatty streaks are very common—they will be found in the arteries of virtually
any middle-aged to elderly person. As such, a fatty streak does not constitute a
problem. However, it tends to progress to more serious stages with time. These
advanced lesions wreak havoc in various ways:
1. Once they become large, they will constrict the lumen of the artery and hence
reduce the blood flow.
2. The endothelium atop an advanced lesion may erode. An intact endothelium
inhibits blood clot formation; endothelial lesions bring the blood into contact
with tissues and molecules that promote blood coagulation. A blood clot, or
thrombus, that forms atop such an eroded lesion will cause acute occlusion
of the artery. This is what happens in myocardial infarction and in most cases
of stroke.
3. An advanced lesion may damage not only the endothelium but also the mus-
cular layer of the arterial wall, which may then rupture. Approximately 20% of
all cases of stroke arise in this manner.
Atherosclerosis is a systemic disease. While most commonly manifest in the
heart and the brain, vascular occlusion and infarction can strike anywhere and
192 11 Cholesterol metabolism
everywhere; for example, constriction of arteries in the legs causes leg muscle pain
even under light exercise (walking), which is known as intermittent claudication.4
A B
C D
Notes: This slide shows cross sections of a normal artery (A) and of atherosclerotic
lesions in different stages of advancement (B–D). The normal artery displays inner
and outer layers of connective tissue, stained in dark purple, as well as a strong
intermediate muscular layer that shows up in a lighter shade. The endothelium is
too thin to be discerned at this low power of magnification. The blood clot in the
lumen is a post-mortem artifact.
Panel B shows a higher power view of an early lesion. The bubbly appearance is
due to foam cells, which are macrophages stuffed chock-full with lipids.5 Panel C
shows an advanced lesion with connective tissue proliferation and accumulation of
detritus within the vessel wall; the lumen of the artery is considerably constricted.
Panel D shows an artery that was already almost completely obliterated by a
proliferating lesion that had encompassed the entire circumference; the narrow
residual lumen is blocked by an acutely formed thrombus (stained yellow-brown).
4
The German vernacular name for this condition translates as “window shopping disease”—not
uncommonly, afflicted patients stop before every window display, feigning interest, in order to
disguise their predicament.
5
The appearance of empty “bubbles” inside the foam cells is an effect of the tissue fixation and
staining technique: Organic solvents used in fixation wash out lipids, and most histological dyes
bind to proteins or nucleic acids but not lipids.
11.6 Cholesterol and atherosclerosis 193
Thrombocyte
A B C D
Notes: With macrophages as with other cell types, uptake of native LDL via the LDL
receptor is regulated by negative feedback, which lets the cells avoid cholesterol
overload. The downregulation of the LDL receptor is achieved by transcriptional
regulation via the SREBP/SCAP pathway (section 11.3).
In contrast to other cells, however, macrophages also have so-called scavenger
receptors, through which they bind and ingest various kinds of debris. Scavenger
receptors do not take up native LDL and are not subject to cholesterol-dependent
regulation. Oxidized or otherwise modified LDL, however, does enter via the scav-
enger receptors, which induces cholesterol overload and transforms the macro-
phages to foam cells. This is a crucial step in the pathogenesis of atherosclerosis.
11.6.7 Experimental modifications that turn LDL into a ligand for the scav-
enger receptor
Notes: In order to better understand what processes might turn LDL into a scav-
enger receptor ligand, various types of chemical modifications have been applied
in vitro. All of the experimental modifications shown here affect the amino groups
of the protein component of LDL (apolipoprotein B). They do not mimic oxidation;
therefore, these findings show that LDL oxidation is not the only mechanism that
may cause pathologically increased uptake of cholesterol into macrophages. Some,
but not all of these modifications are likely to occur in vivo.
11.6 Cholesterol and atherosclerosis 195
NH2 unmodified amine (native LDL)
protein
H
N acetylated amine
protein
O
H
N NH2 carbamylated amine
protein
O
OH OH
N glucosylated amine
protein OH
OH OH
Notes: Oxidized LDL has been demonstrated inside atherosclerotic lesions. Ox-
idation is widely considered to be the most important single mechanism of LDL
modification. It is, however, not very well characterized in molecular terms, and
the relative importance of protein and lipid oxidation is not clear.
R1 R1 R1 R2 R1
HOO• O2
H • O O• O OH
HOOH
R2 R2 R2 R2
peroxidation that requires nothing more than further unsaturated fatty acids and
molecular oxygen.
The peroxidation can also begin with lipid peroxy radicals that are formed
by lipoxygenases, which produce such molecules as precursors of leukotrienes
and other mediators of inflammation. Lipoxygenase inhibitors have shown some
potential to slow down atherosclerosis in animal models; however, it is difficult
to determine how much of that benefit is due to reduced oxidation of LDL, as
opposed to the inhibited signaling by proinflammatory mediators.
The same process of lipid peroxidation also occurs in cell membranes. This
is important in various diseases. An example is favism, a condition in which the
failure to neutralize reactive oxygen species causes the destruction of red blood
cells (see slide 9.4.2).
11.13 How does autocatalytic lipid peroxidation occur, and where does it matter in the
pathogenesis of atherosclerosis?
O O• HO R1
O
R2
•
R1
R2
O
O OH O
R1
R2
O
R2
Notes: Various antioxidants can intercept the autocatalytic process of lipid per-
oxidation. Particularly effective are α-tocopherol and related compounds, which
collectively are referred to as vitamin E, and which due to their fat-soluble nature
are enriched in cell membranes.
You may have noticed that generous amounts of vitamin E are routinely added
to vegetable oil and lard. This is not done out of concern for your health; rather,
the rationale is to protect these foods from turning rancid through peroxidation
of their unsaturated fatty acids.
198 11 Cholesterol metabolism
6
Of note, Adams died at 49, only a few years after finishing his book “The Hitchhiker’s Guide to
the Galaxy,” in which he exposed the far-ranging conspiracy of mice. It would seem that the mice
exacted their revenge!
11.7 Cholesterol metabolism and the treatment of atherosclerosis 199
Mevastatin Atorvastatin
O O HN O
O
N
HO O
HO
HO OH F
OH
O
HO OH
O
Mevalonate
The β-lactam ring in the center of the structure is likely reactive, which suggests
that ezetimibe may bind its target covalently, but I have not found experimental
evidence supporting this assumption.
ezetimibe sitosterol
O
F N OH
F
F F
HO F F
F
OH O NH
S CP-113,818
O N O
N
N N
H H
S S lomitapide
O
LDL
S
cholesterol
CETP triacylglycerol
esters NH
O
HDL
cholesterol
dalcetrapib
Notes: CETP, a serum protein, facilitates the exchange of triacylglycerol and chol-
esterol esters between HDL and LDL. The net effect seems to be an increase of LDL
cholesterol, and accordingly inhibition of CETP looks like a promising strategy.
Dalcetrapib is one of several CETP inhibitors that have been or currently are
under study by various pharmaceutical companies. While dalcetrapib indeed
raises the ratio of HDL cholesterol to LDL cholesterol, large scale clinical trials
have been put on hold due to lack of improvement in clinical outcomes [68].
11.7 Cholesterol metabolism and the treatment of atherosclerosis 201
⊕ ⊖O
N
O
OH
⊕
N
HO OH
Notes: Cholestyramine and similar polymers adsorb bile acids due to a combina-
tion of electrostatic and hydrophobic forces. This prevents the bile acids from
being taken up at the end of the small intestine. They are replaced by de novo
synthesis from cholesterol, which therefore depletes the pool of cholesterol in the
liver.
The strategy is effective but has some side effects. The concentration of bile
acids in the bile is reduced; this promotes precipitation of cholesterol and other
poorly soluble bile constituents, which may then form gallstones. The polymer
particles may also bind some drugs or fat-soluble vitamins and prevent their
absorption.
11.7.7 More . . .
• guar gum and other carbohydrate fibers —absorb and prevent intestinal up-
take of cholesterol and bile acids with variable efficiency
• thyroid hormone analogs—promote LDL utilization
Notes: We will not cover these drugs in detail, but you may find it interesting to
look them up in the literature yourself.
11.14 Explain the major therapeutic strategies for lowering LDL cholesterol.
chylomicrons
LPL
LPL
chylomicron remnants VLDL LDL
LDL receptor
cholesterol
HDL
LCAT ABCA1
cholesterol
nascent HDL
chylomicrons
LPL
LPL
chylomicron remnants VLDL LDL
LDL receptor
cholesterol
HDL
LCAT ABCA1
cholesterol
nascent HDL
Notes: Tangier disease, which is very rare, is interesting from a mechanistic point
of view. The defect concerns the ABC transporter that exports surplus choleste-
rol from the cell for delivery to HDL. As a consequence, HDL is greatly reduced.
Interestingly, LDL cholesterol is also reduced; this may be due to the transfer of
cholesterol between LDL and HDL mediated by CETP (see slide 11.7.4).
Fatty deposits are found in the liver, spleen and cornea. Patients develop
atherosclerosis, type 2 diabetes, and neuropathy.
11.13: The reaction is initiated by superoxide, which abstracts an electron from a double
bond in an unsaturated fatty acid. The fatty acyl radical produced reacts with molecu-
lar oxygen, which produces a peroxy radical that can engage in the same reaction as
superoxide and thus perpetuate the cycle.
Lipid peroxidation can affect LDL and contribute to its oxidation. Oxidized LDL is
taken up by macrophages in atherosclerotic lesions and induces their transformation foam
cells.
11.14: 1. Inhibition of HMG-CoA reductase, the rate-limiting step of cholesterol synthesis
2. Depletion of bile acids with polymer particles (e.g. cholestyramine) 3. Inhibition of
intestinal cholesterol uptake, with either plant sterols (sitosterol) or ezetimibe (inhibitor
of NPC1L1)
11.15: This condition is due to a genetic defect in the LDL receptor. The excessively high
LDL promotes atherosclerosis, which becomes manifest early and severely.
Chapter 12
207
208 12 Amino acid metabolism
• The liver is the major site of degradation for most amino acids, but muscle
and kidney dominate the degradation of specific ones
• Nitrogen is removed from the carbon skeleton and transferred to α-ketoglu-
tarate, which yields glutamate
• The carbon skeletons are converted to intermediates of the mainstream carbon
oxidation pathways via specific adapter pathways
• Surplus nitrogen is removed from glutamate, incorporated into urea, and
excreted
Notes: All amino acids contain at least one nitrogen atom, which forms their α-
amino group; several amino acids contain additional nitrogen atoms in their side
chains. Some nitrogen is used in biosynthesis, for example of nucleotides, but
most of it is surplus and must be eliminated. To this end, the liver incorporates it
into urea, which is released into the bloodstream and excreted by the kidneys.
Removal of nitrogen is typically an early step in the degradation and leaves
behind the carbon skeleton. The structure of the latter is different for each amino
acid, and accordingly each amino acid has its own specific pathway of degradation.
ketogenic
Leu, Lys, Phe, Trp, Tyr acetoacetate acetyl-CoA
Notes: The degradative pathways can be divided into two major classes. As shown
here, most amino acids are converted to intermediates of the citric acid cycle or
to pyruvate, which in turn can serve as precursors for gluconeogenesis; these are
12.2 Transamination of amino acids 209
the glucogenic amino acids. Those amino acids that yield acetoacetate are called
ketogenic, since acetoacetate is one of the ketone bodies (see slide 10.4).
If you look carefully, you will see that phenylalanine and tyrosine are found on
both sides of the divide, since they yield both fumarate and acetoacetate. I suppose
that makes them both glucogenic and ketogenic, although some might insist that
either category should take precedence; this is merely a matter of definition.
Depending on the composition of our diet, amino acids may be very important
as a source of energy. While plant-derived foodstuffs are typically rich in starch,
meat is high in protein but low in carbohydrates. Therefore, when on a diet that
contains mostly meat, amino acids become our major source of glucose.
What happens if protein is supplied in excess of the amount needed to cover
our requirement for glucose? While the liver in principle contains all enzyme
activities required to oxidize the surplus carbon, measurements of the liver’s
overall oxygen consumption indicate that most of the oxidation must happen
elsewhere [71]. Presumably, much of the carbon still ends up as glucose, which
is then either stored as glycogen or released into the circulation. In the latter
case, the surplus will mostly be converted to fat in adipose tissue. A surplus of
ketogenic amino acids will mostly be converted to fat in the liver, which is then
packaged and released as VLDL (see slide 11.4.2).
12.1 Explain why we need a minimum amount of protein in the diet. Also explain the
difference between glucogenic and ketogenic amino acids.
H2 N CH C O C O H2 N CH
CH2 CH2
COOH COOH
Notes: Most standard amino acids lose their α-amino group early on in degrada-
tion through transamination, that is, transfer to an α-keto acid. This is illustrated
here for alanine, which transfers its amino group to α-ketoglutarate to become
pyruvate.2 Transamination is mediated by several different aminotransferase
enzymes. These may be specific for individual amino acids, or they may be able to
process a group of chemically similar ones. The latter applies to the group of the
branched-chain amino acids, which comprises leucine, isoleucine, and valine.
In the case of alanine, the α-keto acid that accepts the amino group is α-
ketoglutarate; this also applies to most other amino acids. Transamination is
freely reversible; therefore, both glutamate and α-ketoglutarate are substrates of
2
Since we already know how to degrade pyruvate, transamination is all that is required to account
for the degradation of alanine. Similarly, as we had seen in slide 6.9.2, aspartate is transaminated to
oxaloacetate, which again suffices to account for its utilization.
210 12 Amino acid metabolism
N H⊕ N
O H ⊕ CH H ⊕ CH
CH H2 O
⊖O ⊖O
⊖O
P P
P
⊕
⊕
N N
N H H
H
O H
O
H3 C C COOH
H3 C C COOH
H3 N⊕ H⊕ N⊕
CH H H CH
⊖O ⊖O
P P
N N
H H
Notes: In this scheme, we again show alanine as the example substrate, but the
mechanism is very general. All transaminases employ the coenzyme pyridoxal
phosphate (PLP), which is the centerpiece of the reaction mechanism. The aldehyde
group of PLP can form an aldimine, or Schiff base, with the α amino group of the
substrate, which releases water.3
After binding of the substrate to PLP, a catalytic base in the active side ab-
stracts its α hydrogen as a proton, and the surplus electron left behind travels all
the way down to the nitrogen at the bottom of the PLP ring, inverting the entire
sequence of single and double bonds along the way. This has the effect of turning
the bond between the α carbon and the α nitrogen into a Schiff base, which then
undergoes hydrolysis to release pyruvate.
At this point, the reaction is only half complete—the nitrogen is still attached
to PLP and needs to be transferred to α-ketoglutarate. The sequence of steps
involved in this transfer is the exact reversal of the ones shown here, so we won’t
show them in detail. However, you might still find it a useful exercise to draw
these reaction steps yourself.
It is interesting to compare the transaminase mechanism with that of serine
hydroxymethyltransferase (slide 15.2.4). While that enzyme breaks the bond be-
tween the α carbon and the side chain rather than the amino group, it uses PLP in
3
At the outset, PLP actually forms a Schiff base with a lysine side chain of the enzyme, which is
then displaced by the incoming substrate; these steps have been skipped here for simplicity. The
liberated lysine then assumes the role of the catalytic base, which is represented here by B.
12.3 Nitrogen disposal and excretion 211
O H2 N O
CH CH CH
⊖O ⊖O ⊖O
P P P
⊕ ⊕
N N N
H H H
Notes: The interaction of the transaminase with its substrates involves four dis-
crete steps: 1) the first substrate enters, 2) the first product leaves, 3) the second
substrate enters, and 4) the second product leaves. Such a strictly sequential order
is referred to as a ping pong mechanism. A ping pong bi bi mechanism is one
that, on top of being strictly sequential, involves exactly two substrates and two
products.4
While two different substrates must be used for the reaction to have some
useful effect, it is of course possible for R1 and R2 to be identical—the reaction
will work just fine, but simply achieve no net turnover.
Notes: While transamination solves the problem of removing the α-nitrogen for
the amino acids other than glutamate, there also must be mechanisms for regen-
erating the α-ketoglutarate that is converted to glutamate in each transamination
reaction, and for the ultimate disposal of nitrogen. A reaction that directly regen-
erates α-ketoglutarate is catalyzed by glutamate dehydrogenase, as follows:
HO O− HO O P O− −O NH2 −O P O NH2
O− O
NH3
Notes: The urea cycle runs only in the liver. It begins with the incorporation of am-
monia into carbamoylphosphate by the corresponding synthetase. This reaction
occurs in three successive steps. The first step uses ATP to activate bicarbonate
to carbonylphosphate, which then captures free ammonia to form carbamate. An-
other ATP-dependent step activates that intermediate to carbamoylphosphate. The
carbamoyl group will find its way into the urea that is produced by the urea cycle.
5
In liver cirrhosis, one of the main problems is the lacking capability of the liver to detoxify am-
monia derived from bacterial metabolism in the large intestine. Oral antibiotics (e.g. paromomycin)
are used in this condition to reduce bacterial growth and ammonia formation.
Some ammonia is excreted with the urine as well, where it serves to buffer surplus protons also
destined for excretion. However, this ammonia is not extracted from the circulation but is formed
from glutamine directly in the kidneys.
12.3 Nitrogen disposal and excretion 213
NH2 carbamoyl- P
citrulline
O O P
NH2 ATP aspartate
NH
2 3
NH2 O NH AMP
O NH
Pi PPi HOOC
NH2 NH2
NH2 NH2
ornithine
COOH COOH HOOC
COOH
H2 O
3
5
NH2 AMP
4
NH HOOC NH
H2 N O
arginino-
H2 N NH N NH
urea H succinate
HOOC
HOOC
NH2 NH2
arginine fumarate
COOH HOOC COOH
Notes: To answer this question, we just need to pull together our previous knowl-
edge about transamination as well as the citric acid cycle. Fumarate is turned into
malate and then oxaloacetate in the citric acid cycle, so we can just borrow those
reactions. Oxaloacetate can be transaminated by aspartate aminotransferase us-
214 12 Amino acid metabolism
ing glutamate (slide 6.9.2), which in turn acquired its nitrogen by transamination
of some other amino acid destined for degradation. In other words, the aspartate
simply serves as an intermediate carrier of nitrogen en route from amino acid
degradation to urea synthesis.
HCO–3 , ATP
NH3 Cit
Asp
oxaloacetate
Orn AS
urea cycle
malate
transaminases
glutamate dehydrogenase Arg fumarate
TCA cycle
urea
The network of reactions shown in this slide accounts for the disposal of nitrogen
that accrues in amino acid degradation in the liver. As stated at the outset, other
tissues also break down amino acids; for example, skeletal muscle metabolizes the
lion’s share of the branched-chain amino acids. Therefore, a mechanism is needed
to ferry the nitrogen produced in the peripheral organs to the liver. Ammonia
cannot be used as a carrier, since it is too toxic; amino acids are a better alternative.
The two most important nitrogen carriers are alanine and glutamine (see below).
mitochondria cytosol
2 ADP fumarate
carbamoyl-
phosphate H+ H+ ATP AMP
aspartate
are exchanged for each other by a specific transporter in the inner mitochondrial
membrane.
Ornithine has two free amino groups, while citrulline has one. The exchange
transport is rendered electrically neutral by the cotransport of a proton out of the
mitochondria, that is, against its concentration gradient. The energetic cost of this
uphill transport is offset by the expenditure of ATP in other steps of the urea cycle.
Nevertheless, the coupling of this substrate exchange to proton export will keep
the cytosolic concentration of citrulline low at equilibrium.
Also note that the reactions that involve fumarate and aspartate occur in the
cytosol. We had just noted that the conversion of fumarate back to aspartate
involves some reactions borrowed from the TCA cycle. That cycle runs in the
mitochondria; however, fumarate does not need to enter the mitochondria at this
stage, since all required enzyme activities are also present in the cytosol.
1 1 1 2
glucose glucose
Notes: Glutamine is the most abundant amino acid in the blood; it is significant
both as a nitrogen and a carbon carrier.6 It can bring about a net transfer of nitro-
gen from peripheral tissues to the liver in exchange for glutamate. The enzymes
6
Some cell types, including leukocytes and the intestinal epithelia, use glutamine rather than
glucose as their major energy-providing substrate.
216 12 Amino acid metabolism
involved in the overall scheme are transaminases (1), glutamate dehydrogenase (2),
glutamine synthetase (3), and glutaminase (4). The latter two reactions are shown
in detail in slide 12.3.7.
1 2 3 4
One might reason that, in the liver, glutamate could be further deaminated by
glutamate dehydrogenase, and α-ketoglutarate be returned to the periphery, which
would allow the transfer of two nitrogen atoms in each turn of the cycle. This
should work in principle, but the plasma concentration of α-ketoglutarate is too
low for it to be quantitatively important.
12.4 Explain how nitrogen that accrues in the degradation of amino acids in muscle tissue
is transported to the liver.
glutamine
NH2 glutaminase
synthetase
O
oxaloacetate aspartate
it to aspartate in order to feed the urea cycle. Together with glutamine, it also
controls the level of free ammonia and accomplishes the transport of nitrogen
between organs.
As shown in this scheme, glutamate is formed from glutamine by glutaminase,
and it can be turned back into glutamine by glutamine synthetase.7 Evidently, both
enzymes together would create a futile cycle that would accomplish nothing except
ATP hydrolysis. In most organs, only one or the other enzyme has significant
activity; for example, glutamine synthetase predominates in skeletal muscle (see
slide 12.3.6), whereas glutaminase is abundant in the kidneys, which use it to
secrete ammonium chloride into the urine when eliminating excess acid.
The liver contains both glutaminase and glutamine synthetase, which would
suggest that futile cycling should occur. However, as it turns out, the enzymes are
present inside the same tissue but not the same cells. Instead, they are distributed
strategically within the liver lobule so as to create a confined compartment to host
the urea cycle (see next slide).
Notes: The toxicity of ammonia mandates that its concentration be kept very
low in the systemic circulation. On the other hand, for the urea cycle to run
at speed, the concentration must be high enough to saturate the initial enzyme,
carbamoylphosphate synthetase, to a useful degree. Therefore, ammonia must be
released when the blood enters the liver tissue, and scooped up again before the
blood is drained away into the general circulation.
To make this work, the enzymes that release or fix ammonia, respectively,
are strategically distributed in the liver tissue. We had seen before that the liver
consists of functional units called lobules, with the blood filtering through each
lobule from the periphery towards the center, from where it is drained toward
the systemic circulation (slide 1.6.3). Glutaminase and glutamate dehydrogenase,
which release ammonia, are found predominantly in the periphery of the lobule,
7
The reaction mechanism of glutamine synthetase is shown in slide 2.4.1.
218 12 Amino acid metabolism
the so-called periportal zone. Here, they increase the concentration of free ammo-
nia, allowing the urea cycle to run at speed. As the blood seeps into the pericentral
zone toward the lobule’s central vein, ammonia is scavenged again by glutamine
synthetase before the blood is drained from the liver into the general circulation.
A similar spatial separation applies to the enzymes of arginine degradation.
The first step in this pathway is catalyzed by arginase, which also functions in the
urea cycle and produces ornithine. In the periportal zone, it would be deleterious
to continue the degradation beyond ornithine, since this would drain the urea
cycle of its intermediates. Therefore, the next enzyme in the pathway, ornithine
aminotransferase, is only found in the periportal zone, in which the urea cycle
must shut down anyway.
The purposeful distribution of different enzyme activities illustrates nicely
how biochemical and anatomical levels of organization are interrelated, and how
our body is not just a bag of cells, not even at the level of individual organs and
tissues.
glutamine
NH3
glutamate
oxaloacetate N-acetyl-glutamate
α-ketoglutarate carbamoyl- P
aspartate ornithine
urea
Notes: At the heart of the mechanisms that control flow through the urea cycle,
we once again find glutamine and glutamate. Glutaminase gets the ball rolling by
releasing ammonia from glutamine. Quite unusually, ammonia exercises positive
feedback on glutaminase, which causes a rapid accumulation of both glutamate
and ammonia. High levels of glutamate then promote the incorporation of ammo-
nia into urea in several ways.
1. Some glutamate is converted to N-acetylglutamate. This is a regulatory
molecule that allosterically activates carbamoylphosphate synthetase.
2. Glutamate is also the biosynthetic precursor of ornithine. A high level of
glutamate will also raise the level of ornithine, which will increase the flow
through the urea cycle.
12.4 Degradative pathways of individual amino acids 219
3. As we have seen, glutamate itself feeds nitrogen into urea synthesis via gluta-
mate dehydrogenase or via aspartate.
Note that all these regulatory events amplify the flow through the urea cycle;
they are the ones that run in the periportal zone of the liver lobule. As stated
above, the urea cycle is shut down in the pericentral zone through the capture of
remaining ammonia by glutamine synthetase as well as by ornithine degradation.
12.5 Explain how flow through the urea cycle is controlled within the liver lobule.
Notes: Urea cycle defects primarily become symptomatic due to the accumulation
of ammonia, which impairs brain function. Another aspect is the deficiency of argi-
nine. In healthy individuals, arginine can be diverted from the urea cycle toward
protein synthesis as required, but this supply is lacking if the cycle is disrupted.
This may induce protein catabolism, thereby exacerbating the symptoms. The
problem is addressed by the addition of arginine to the diet. If the enzyme defect
is not between citrulline and arginine, it is possible to supply citrulline instead;
this has the advantage of picking up one nitrogen equivalent en route to arginine.
The rationale for treating these enzyme defects with a protein-restricted diet is
fairly obvious. Another, more intriguing approach is known as alternate pathway
therapy. Here, the patients are given several innocuous organic acids that are
substrates for conjugation with amino acids. These conjugates then serve as an
alternative vehicle for the renal elimination of surplus nitrogen. This ingenious
form of treatment is further discussed in slide 18.3.7.
12.6 Explain the pathogenesis and treatment of urea cycle enzyme defects.
The degradation pathways for the individual amino acids vary considerably in
complexity. As we had seen, some amino acids only require a single transamination
step; on the other hand, others have lengthy degradation pathways with intriguing
catalytic mechanisms. We will here consider some selected examples; several
others are discussed in a later chapter (slides 15.2.4–15.2.7).
220 12 Amino acid metabolism
HO H2 O HO α-KG HO
O O O
1 2
H2 N H2 N O
O NH2 O Glu O
NH2 OH OH
HO HO H2 O HO
O O O
H2 N H2 N O
HO H2 O NH3
Notes: Serine, another non-essential amino acid, can be degraded along several dif-
ferent pathways; this slide shows one of them. Only the first step is enzymatically
catalyzed; the aminoacrylate produced is unstable and spontaneously hydrolyzes
to pyruvate. The second step releases ammonia, which must be disposed of. It
seems that in humans the reaction occurs only in the liver, where the ammonia
can directly enter the urea cycle.
Like the transaminases, the enzyme uses pyridoxal phosphate, and the role
of the coenzyme is often presented along the lines of the usual electron sink
8
The enzyme used for this treatment is purified from E. coli. In healthy patients, repeated injec-
tions of a bacterial protein would soon induce antibodies, which would quickly render the enzyme
inactive. However, in leukemia patients, the disease itself, and the cytotoxic drugs simultaneously
applied—such as, for example, cytosine arabinoside (slide 16.9.7)—conspire to suppress antibody
formation. If it occurs anyway, enzyme prepared from another bacterium, Erwinia chrysanthemi,
can be used. Immunogenicity of the enzyme can be reduced by derivatization of the protein with
polyethyleneglycol (PEG).
12.4 Degradative pathways of individual amino acids 221
mechanism (see slide 12.2.1). However, based on the crystal structure of the
enzyme, a different mechanism has been proposed, in which no electron sink
appears and instead the phosphate group of PLP plays a prominent role [72]. I am
not enough of a chemist to judge how plausible this mechanism may be.
Notes: Leucine, isoleucine and valine are collectively referred to as the branched-
chain amino acids. Unlike the other amino acids, these ones undergo degradation
mostly in skeletal muscle. This is reminiscent of fatty acids, which are also de-
graded prominently in muscle, and indeed several steps in leucine degradation
have similarity with the reactions we have seen in fatty acid metabolism. Leucine
degradation involves the following steps:
1. Transamination by branched chain amino acid (BCAA) transaminase yields
α-ketoisocaproate.
2. α-Ketoisocaproate is decarboxylated and dehydrogenated by branched chain
α-keto acid dehydrogenase. Like the transaminase in step 1, this dehydroge-
nase participates in the degradation of all branched chain amino acids (valine,
leucine, isoleucine). The reaction mechanisms and the structural organization
222 12 Amino acid metabolism
HO HO
α-KG CoA-SH CO2 FAD FADH2
O O S CoA
H2 N O O
1 2 3
+ +
Glu NAD NADH+H
S CoA
O
S CoA
S CoA S CoA
O
O H2 O O CO2
O 6 5 4
O O
O HO
ADP+Pi ATP
HO HO
HO
HO O HO O HO O
O2 + BH4 α-Ketoglutarate O2
HO O
H2 N H2 N O OH
1 2 3
OH OH OH
O2
Fumarylacetoacetate 4
O O O O HO O
O
O HO O
O
OH 5
N O O
O H2 O O
6
OH
F F O O O
F O Maleylacetoacetate
HO HO
OH
NTBC Acetoacetate Fumarate
Since there are so many different pathways for the degradation of the various
amino acids, it is understandable that many of the known inborn errors of metabo-
lism are related to amino acid metabolism. We will consider a few examples that
affect the pathways discussed here.
10
As you may have guessed, that full name was a straight copy-and-paste job.
224 12 Amino acid metabolism
challenge, then, is to diagnose the disease in newborn kids, before any damage
is done. Happily, the enzyme defect does not cause a problem during fetal de-
velopment, since the placenta constantly equilibrates both useful and potentially
harmful metabolites between the maternal and the fetal circulation. Buildup of a
metabolite in the fetus will therefore not occur as long as the mother’s metabolism
is able to degrade it.
The modern test for phenylketonuria is effective but boring—a sample of
blood is drawn, and the phenylalanine concentration in the serum is determined by
HPLC. The original test—the Guthrie test—was a bit more roundabout in principle,
yet ingenious and exceedingly simple and cheap in practice. Moreover, it well
illustrates the power of bacterial genetics in biochemistry, and it therefore merits
discussion here.
In contrast to mammalian cells, the bacterium Escherichia coli can synthesize
all 20 standard amino acids, as long as it has ammonia, some inorganic salts, and
an organic carbon source such as glucose. Such a substrate mixture constitutes
a minimal medium. The Guthrie test makes use of a mutant E. coli strain that is
Phe– , which means that it is unable to synthesize phenylalanine on its own. This
strain can be grown on a rich medium that supplies phenylalanine; however, when
spread onto minimal medium, it will not grow.
Now, if we take a little snippet of filter paper soaked with a drop of baby
blood and place it on top of the inoculated minimal medium, any phenylalanine
contained in it will diffuse into the surrounding agar. If there is enough of it in the
sample, this will allow the bacteria in the vicinity to resume growth. Therefore, a
zone of bacterial growth surrounding a blood sample will identify a patient with
phenylketonuria. Brilliant!
Note that, for this test to work, we cannot collect the blood sample right away
after birth. As noted above, the fetal blood equilibrates with the mother’s, and so
the phenylalanine concentration is only slightly increased at birth. We therefore
must allow 1–2 weeks after delivery for phenylalanine to accumulate in the child’s
blood for the Guthrie test to respond. This is a drawback of the test relative to
the HPLC method—the latter is more quantitatively accurate and readily detects
the smaller increase in phenylalanine concentration that is present at the time of
delivery.
Notes: The variation of the gene frequency for PKU between races and geograph-
ical areas suggests that some regional environmental conditions may confer a
selective advantage to the heterozygous state, as is the case with sickle cell ane-
mia and glucose-6-phosphate dehydrogenase deficiency in regions with endemic
malaria. It has been proposed that the heterozygote advantage in PKU consists
in protection from the fungal toxin ochratoxin A, which is produced by some
Aspergillus molds that cause food to rot [74].
Ochratoxin A competitively inhibits the coupling of phenylalanine to its cog-
nate tRNA by the corresponding aminoacyl transferase and thereby disrupts pro-
226 12 Amino acid metabolism
tein synthesis. It is more toxic to fetuses than to adults, most likely because
fetuses are short of the enzymes that inactivate xenobiotics and toxins such as
ochratoxin. Mothers who are heterozygous for PKU will have a somewhat higher
level of phenylalanine, which will be shared with the fetus via the placenta. This
will counter the inhibition of tRNA aminoacylation in the fetus and thereby afford
it some measure of protection.
Phe-tRNA synthetase
OH
OH
NH2
OH Cl O
NH2 OH
O H
N O
O
O OH O
One of the places with the highest abundance of PKU is Ireland. This country is
also known for its repeated historic episodes of severe famine. Starving people will
tend to eat rotten food rather than discard it. Indeed, reference [74] reports that
lowered rates of abortion were found in Irish women who were heterozygous for
PKU. I have not ascertained whether the time periods covered by those statistics
coincided with periods of actual famine.
12.10 Explain the cause, pathogenesis, diagnosis and treatment of phenylketonuria.
12.6 Answers to practice questions 227
12.5.4 Tyrosinemia
12.7: Some types of leukemic cells lack the capacity to synthesize asparagine and are
therefore susceptible to its depletion in the blood by asparaginase. Normal cells are not
affected by this treatment.
12.8: 1) Serine dehydratase induces deamination of serine to pyruvate, 2) serine-pyru-
vate transaminase produces glycerate, and 3) serine hydroxymethyltransferase produces
glycine and N,N ′-methylenetetrahydrofolate.
12.9: The degradation pathway begins with transamination and a subsequent step that
resembles pyruvate dehydrogenase. The resulting acyl-CoA undergoes FAD-dependent
β-dehydrogenation, carboxylation and hydration; this produces HMG-CoA, which in turn
yields acetoacetate and acetyl-CoA.
12.10: PKU is due to a homozygous enzyme defect for phenylalanine hydroxylase. Phe-
nylalanine builds up and dislodges tryptophan from the shared transporter that mediates
uptake of both amino acids into the brain, which causes a lack of tryptophan and hence
serotonin in the brain. The disease can be diagnosed by direct measurement of phe-
nylalanine plasma levels or through the Guthrie test, which a phenylalanine-auxotrophic
bacterial strain. Treatment consists in a phenylalanine-restricted diet.
12.11: Tyrosinemia is due to a homozygous defect of fumarylacetoacetate hydrolase.
This causes the build-up of fumarylacetoacetate and maleylacetoacetate, which are chemi-
cally reactive and cytotoxic. Treatment uses dietary restriction and NTCB, which inhibits
p-hydroxyphenylpyruvate dioxygenase and thereby forestalls the build-up of the toxic
metabolites downstream.
Chapter 13
Hormone Message
Notes: We have already touched briefly on the effects of insulin, glucagon and
epinephrine on gluconeogenesis and glycogen metabolism. In this chapter, we
will take a more thorough look at these hormones’ properties and activities. This
will provide the foundation for the next chapter, in which we will consider the
disruptions of metabolic regulation that occur in diabetes mellitus.
Of the hormones listed in the table, only insulin has the effect of lowering
blood glucose. The other hormones are all antagonistic to insulin, and a pathologi-
cal increase in their secretion may result in symptomatic diabetes. In the case of
glucocorticoids, symptomatic diabetes may also arise from their use as drugs.
Both glucocorticoids and thyroid hormones cause their effects mostly through
up- and down-regulating gene transcription. The two types of hormones control
different sets of target genes, but both upregulate the expression of β-adrenergic
receptors, that is, receptors for epinephrine, and so amplify the metabolic effects
of this hormone.
13.1 Which hormones are antagonistic to insulin with respect to the blood glucose level?
229
230 13 Hormonal regulation of metabolism
13.2 Insulin
Notes: Insulin is produced in the islets of Langerhans, which are small clusters
of cells that occur interspersed within the exocrine pancreas. In this picture, one
islet is shown in the center, and a tangential section across another is seen in the
bottom right. The two islets are embedded in a “red sea” of exocrine pancreas
tissue.
The function of the exocrine pancreas—secretion of digestive enzymes and
of bicarbonate into the small intestine—has been discussed in slide 1.6.7. The
products of islet cells are secreted into the bloodstream; therefore, the islets
collectively function as an endocrine gland. Among the several cell types found in
the islets, the β-cells produce insulin, whereas the α-cells produce glucagon.
Notes: When purifying a protein of interest from some organ, standard practice is
to first grind up the tissue with a homogenizer, and then to subject the homogenate
to various fractionation procedures so as to incrementally enrich the protein,
13.2 Insulin 231
apply ligature,
reseal abdomen
obtain protease-free
homogenized extract
Notes: The bright idea that overcame this problem occurred, in one of his sleepless
nights, to a young physician named Frederick Banting. He immediately resolved
to pursue this idea at the University of Toronto, where he was joined in this effort
by a junior colleague, Charles Best.
It had previously been observed in human patients that occlusion of the pan-
creatic duct induced a protracted self-destruction of the exocrine pancreas, while
sparing the pancreatic islets. Banting surgically obstructed the pancreatic ducts of
experimental animals to induce destruction of the exocrine pancreas. The source
of the contaminating proteases was thus removed, and Banting and his colleagues
were able to extract and purify insulin from the islets that remained intact within
the biochemically inert scar tissue that had replaced the exocrine pancreas.
It is worth noting that Banting and his colleagues did not proceed to claim a
patent, but instead shared their discovery freely, encouraging everyone else to use
it. Their generosity ensured that the diabetics of the world were soon supplied
with insulin.
13.2 How did Banting solve the problem of proteolytic degradation of insulin in pancreas
extract?
1
In modern practice, one could likely prevent this using protease inhibitor cocktails; however,
such inhibitors were not available at the time.
232 13 Hormonal regulation of metabolism
“Comrade Bethune’s spirit, his utter devotion to others without any thought of self, was
shown in his great sense of responsibility in his work and his great warmheartedness
towards all comrades and the people. Every Communist must learn from him.”
Mao Zedong, “In Memory of Norman Bethune”
Notes: Of historical interest, but not relevant to the main subject, is that one of
Banting’s classmates at medical school was Norman Bethune, a gifted surgeon
who invented many surgical instruments and introduced mobile blood transfusion
units in the Spanish civil war. He later accompanied Mao Zedong and his guerrilla
fighters on their Long March across China, a long and costly, but ultimately suc-
cessful retreat from the advancing Kuomintang that occurred during the civil war
of the 1930s.
The picture on the left shows Bethune performing surgery in a makeshift oper-
ation room during the Long March; during one such operation, Bethune contracted
a bacterial infection to which he subsequently succumbed. The picture on the
right shows Bethune’s birthplace in the city of Gravenhurst, which is located in
Ontario’s Muskoka region. It is now a museum and is well worth a visit.
13.3 When was the Spanish Civil War?
G
I
V
E Q C C T S I C S L Y Q L E N Y C N T
K
P
T
Y
F
F
F V N Q H L C G S H L V E A L Y L V C G E R G
Notes: The insulin molecule consists of two peptide chains, which are held to-
gether by two disulfide bridges. The sequence of insulin was the very first protein
sequence ever to be determined, and the methodology for this feat was developed
by the gentleman with the sardonic smile, Frederick Sanger.
13.2 Insulin 233
Notes: Until the late 1980s, swine and cow insulins were the mainstay of insulin
substitution therapy. They are fully active in humans but differ from human insulin
in one or three amino acid positions, respectively. This difference may promote
the formation of antibodies, which bind and inactivate insulin. Recombinantly
234 13 Hormonal regulation of metabolism
expressed human insulin has replaced the animal insulins in therapy, which has
largely done away with this problem.2
GIVEQCC TSICSLYQLENYCN
F V N Q H L C GSHLVEALYLVCGERGFFYTPKT
GIVEQCC TSICSLYQLENYCN
F V N Q H L C GSHLVEALYLVCGERGFFYTPKA
GIVEQCC ASVCSLYQLENYCN
F V N Q H L C GSHLVEALYLVCGERGFFYTPKA
Notes: While insulin controls the rate of glucose uptake in many tissues, the β-
cells, which control the release of insulin, themselves take up glucose in an insulin-
independent manner; the rate of uptake cells thus simply depends on the plasma
glucose level. Inside the β-cells, glucose undergoes degradation, which increases
the cellular level of ATP. The ATP then binds to the sulfonylurea receptor, which
in turn closes a potassium channel associated with it. This leads to an increase
in the membrane potential, which activates a voltage-gated Ca++ channel. Ca++
entering the cell triggers the exocytosis of insulin and C-peptide.
Glucose is not the only substrate that can be degraded to yield ATP, and it
is not surprising that some amino acids and fatty acids will also promote insulin
secretion. In addition, several types of cell surface receptors contribute to the
activation of insulin secretion in a manner that is not dependent on substrate
degradation.
2
More recently, recombinant insulins with point mutations have been introduced into clinical
treatment. Interestingly, these insulins seem to be less prone to antibody induction than porcine
and bovine insulins were. This may be due to their reduced tendency to form aggregates (see slide
14.5.11).
13.2 Insulin 235
Glucose Insulin
Ca2+ Ca2+
Glucose
Membrane
ADP + Pi Insulin
ATP potential
K+ K+
ATP ATP Sulfonylurea receptor
H2 O, CO2
Notes: This figure illustrates how the polypeptide chains of the sulfonylurea re-
ceptor and of the associated “inward rectifier” Kir channel crisscross the cell mem-
brane. The N-terminus of the sulfonylurea receptor is extracellularly located. NBF1
and NBF2 are nucleotide-binding folds, that is, conserved protein sequence motifs
that are involved in the binding of ATP.
The scheme shows a single Kir molecule; however, a functional K+ channel consists
of four Kir subunits. The entire ensemble of Kir and sulfonylurea receptor subunits
is referred to as a KATP channel.
13.2.10 KATP channels also regulate the tone of smooth muscle cells
Notes: KATP channels serve in more than one physiological role. In vascular
smooth muscle cells, KATP channels regulate the strength of contraction. Sus-
tained contraction of a muscle cell will reduce its ATP level, which will promote
dissociation of ATP from the sulfonylurea receptor and open the connected Kir
channel. The increase in K+ permeability will lower the membrane potential and
inhibit the activation of voltage-gated calcium channels; this, in turn, will inhibit
cell contraction. This mechanism protects the cell from excessive exertion: when
ATP is depleted, the opening of the KATP channels will cause the cell to ignore any
236 13 Hormonal regulation of metabolism
further calcium signals and suspend contraction until it has caught its breath and
replenished ATP.
Drugs that counteract the effect of ATP on the sulfonylurea receptor will keep
the Kir open and promote relaxation of vascular smooth muscle cells. This is an
effective means to lower blood pressure.
HN
N
HN O N
O N H
S NH N
O S O O +
H2 N N NH2
O
O
S
Cl O−
O
Notes: The insulin receptor is found on the surface of all body cells that respond
to the hormone. The highest receptor density is found on liver cells; in fact, more
than half of the insulin that is released by the pancreas is captured by receptors
in the liver (recall that venous blood from the pancreas is drained into the portal
vein; see slide 1.6.2).
The insulin receptor is located in the cytoplasmic membrane; it is a receptor
tyrosine kinase. Aside from insulin, human growth hormone and many other
growth factors have receptors of this type. Receptor tyrosine kinases are one of
the major functional classes of hormone receptors.
A receptor tyrosine kinase has two functional domains. The extracellular
domain binds to the hormone. This causes a conformational change to the entire
receptor, which activates the intracellular protein tyrosine kinase domain. The
activated receptor binds one or several cognate protein substrates, which it then
phosphorylates at specific tyrosine residues. The phosphorylated substrates leave
the receptor and interact with downstream adapter proteins, which then set off
various intracellular signaling cascades.
13.2.13 Insulin receptor first phosphorylates itself and then a number of in-
sulin receptor substrate proteins
Notes: In the case of insulin receptor, the first target for phosphorylation is an-
other insulin receptor molecule; mutual phosphorylation of the two receptors
locks both into the active conformation. Subsequently, the receptor molecules
phosphorylate a series of regulatory proteins, which are referred to as insulin
receptor substrates (IRS).
13.6 What is the signaling mechanism of the insulin receptor?
238 13 Hormonal regulation of metabolism
Insulin also activates phosphodiesterase, which lowers cAMP and thereby also
affects the phosphorylation of glycogen synthase and of phosphorylase kinase
(see slide 8.4.2). This effect is mediated via protein kinase B, too, but the exact
molecular cascade from PKB to phosphodiesterase is not clear.
13.7 How does insulin affect glycogen metabolism?
13.2 Insulin 239
Notes: The brain must keep working at all times; it depends on glucose and can
take it up from the blood with or without insulin. In contrast, most other tissues
can more readily replace glucose with other energy-rich substrates. The uptake of
glucose into the cells of those tissues depends on insulin. When glucose supply is
low, insulin secretion drops as well. Glucose uptake by insulin-dependent tissues
ceases, which preserves glucose for the brain.
If blood glucose drops to excessively low values—this state is called hypo-
glycemia—the brain will no longer manage to obtain enough glucose, which will
lead to unconsciousness and can result in brain damage and death.
Glc
Glc
N′ H S Q G T F T S D Y S K Y L D S R R A Q D F V Q W L M N T C′
Glucagon
HO O
OH H2 N
HN
O
HO
OH
HO
I I
O I
HO
OH O OH
Notes: Glucagon is a peptide hormone that is produced in the α-cells of the pan-
creatic islets. Epinephrine is produced in the medulla, and cortisol in the cortex of
the adrenal glands. All these hormones are antagonists of insulin.
3
The rate of GTP cleavage is modulated by special regulatory proteins, which we will not go into
here. There just seems to be no end to the layers, convolutions and intricacies of signal transduction
in cells; compared to it, metabolism is positively sane, simple and elegant.
242 13 Hormonal regulation of metabolism
13.3.2 The glucagon and epinephrine receptors activate adenylate cyclase and
protein kinase A
Notes: One type of G protein can couple to several types of GPCR; this is the
case with the the so-called stimulatory G protein (GS ), which couples to both the
glucagon receptor and the β-adrenergic receptor, which binds epinephrine. The
α-subunit of this G protein (αS ) activates adenylate cyclase, which converts ATP to
cyclic AMP (cAMP). This second messenger then binds and activates protein kinase
A (PKA), which phosphorylates a number of target enzymes.
Adenylate cyclase is also targeted by the α-subunit an inhibitory G protein, Gi ,
which is activated downstream of other GPCRs, such as for example α2 -adrenergic
receptors or several serotonin receptor subtypes. Another major signaling cas-
cade that is controlled by GPCRs is the phospholipase C/protein kinase C pathway,
which is activated for example by vasopressin and oxytocin receptors. In these
notes, however, we will confine the discussion to epinephrine, glucagon, and pro-
tein kinase A.
Notes: This slide summarizes downstream effects of PKA activation that were
already discussed in detail earlier. The roles of PKA in gluconeogenesis and
in glycogen synthesis are shown in slides 7.5.4 and 8.4.2, respectively. PKA also
activates hormone-sensitive lipase in fat tissue, which induces the release of free
fatty acids and glycerol (slide 10.3.7).
13.3 Other hormones 243
Notes: While they are inactive, nuclear hormone receptors are located in the cy-
tosol. When activated by ligand binding, they translocate to the nucleus and bind
to cognate DNA sequences, recruit a number of other regulatory proteins (not
shown) and ultimately induce the transcription of genes in the vicinity of their
target DNA sequences.
5′ 3′ 5′ 3′ 5′ 3′
3′ 5′
Note that TRβ and RXR bind to the same hexanucleotide motif. Nevertheless,
the two receptor dimers recognize different DNA target sequences, since the two
instances of the hexanucleotide differ in orientation and spacing. Therefore, the
two dimers bind to different sites on the DNA and control different sets of genes.
One key mechanism through which thyroid hormones affect energy metabo-
lism is the transcriptional induction of mitochondrial uncoupling proteins. As
discussed earlier (slide 6.3.1), uncoupling proteins mediate the conversion of me-
tabolic energy to heat and therefore increase the burn rate of glucose and other
energy-rich substrates. Nevertheless, thyroid hormones do not reduce blood glu-
cose, since they also induce the expression of β-adrenergic receptors, and they
therefore amplify the glucose-enhancing effect of epinephrine.
Dexamethasone OH Prednisolone OH
O O
100
Effect (% dexamethasone)
OH OH IL-1β↓
HO HO
75 TAT ↑
F
50
O O
25
N OH O
0
HO
ne 4858 on
e
isolo 2 r ist
U p
Pr edn R
Mif
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O Mifepristone O RU 24858
Diabetes mellitus
14.1 Introduction
Apart from atherosclerosis, diabetes mellitus is the most common metabolic dis-
ease. It is caused by an absolute or relative lack of insulin activity, which disrupts
the regulation and balance of many metabolic pathways. Examining the pathogen-
esis of diabetes is instructive and a good opportunity for us to recapitulate what
we have learned so far.
1
Diabetes insipidus is due to the lack of anti-diuretic hormone (ADH), a peptide secreted from
the posterior hypophyseal gland. This hormone promotes the retention of water by reuptake in the
collecting duct section of the nephron (see below). Loss of ADH production, typically due to a lesion
of the hypophyseal gland, results in a dilute urine, but without loss of glucose or other metabolites.
247
248 14 Diabetes mellitus
Notes: Type 1 diabetes is the form typically observed in the young, whereas the
type 2 is more frequent overall and is typically observed in the elderly. MODY—
maturity type onset diabetes of the young—is type 2 diabetes in young people.
Form Cause
The loss of glucose with the urine is a consequence of too high glucose blood
levels, which result from the failure of uptake and utilization by insulin-dependent
tissues, as well as from various kinds of metabolic dysregulation (see below).
Renal glucose loss is not the most severe symptom of diabetes, but it is never-
theless a prominent one. In order to understand how it comes about, we will first
take a look kidney function. This will also provide useful background for some
topics in later chapters.
2. solute reuptake: glucose, amino acids, salts etc. are recovered from the ultra-
filtrate through active transport
3. water reuptake: driven by osmotic gradient
4. solute secretion: some substrates are actively secreted into the nascent urine
Notes: Blood flows through the kidneys at a rate of ~1.2 l/min, or ~1700 l/day. A bit
more than half of that volume is blood plasma, and if we assume a plasma filtration
rate of 15%, we obtain a filtrate volume of approximately 150 l/day. Obviously,
therefore, most of the water and of the solutes contained in the filtrate must be
reclaimed, and only the leftovers appear in the final urine. On the other hand,
some solutes are secreted into the nascent urine only after filtration.
Macula densa
Glomerulus
Distal
tubule
Proximal
tubule
Collecting duct
Loop of Henle
Notes: Just as the liver consists of many lobules that each comprise the whole
organ function in a small microcosm, the kidney also consists of many instances
of a repeating functional unit, the nephron, that comprises all stages of urine
production. Each of the two kidneys contains ~1.3 million nephrons that all work
in parallel.
A nephron consists of a glomerulus and a tubular part that can be divided
into the proximal tubule, the loop of Henle, and the distal tubule; the latter joins a
collecting duct that drains several nephrons towards the exit.
The primary filtrate is formed in the glomerulus. It flows into the proximal
tubule, where glucose, amino acids, and most of the salt ions are reclaimed by
specific active transporters; water follows by osmosis. More water is reclaimed
in Henle’s loop. Along this stretch, a very high salt concentration prevails in the
interstitial space of the surrounding tissue, which allows the nascent urine to
become significantly more concentrated than blood plasma. Fine tuning of urine
concentration, pH and salt content occurs in the distal tubule.
Reuptake of glucose in the nephron occurs through sodium-coupled active
transport. The number and capacity of the transporter molecules in the proximal
tubules is limited.
250 14 Diabetes mellitus
Notes: The tissue section shows two glomeruli, surrounded by cross sections of
various types of tubules. The sheath containing each glomerulus is known as
Bowman’s capsule. In the glomerulus on the right, the capsule has been cut away
where it opens into the proximal tubule.
Notes: The glomerulus contains a coiled arteriole, that is, a small artery. Unlike
the arterioles in the remainder of the body, the endothelium of these glomerular
arterioles has gaps. The endothelial gaps line up with gaps in a second cellular
layer that is formed by the so-called podocytes and surrounds the outer side of the
basal membrane of the arteriole. As with the capillaries in the general circulation,
it is the basal membrane that forms the actual molecular sieve in the filtration.
The molecular weight cutoff for filtration is similar, too: molecules with less than
10 kDa get across, whereas larger ones are retained. For small molecules that are
not retained in the plasma by protein binding, the concentration in the filtrate
will be the same as in the plasma. In contrast, proteins and other large molecules
should be quantitatively retained inside the arteriole. If proteins do appear in the
urine in significant amounts, this indicates that the filtration apparatus is damaged,
as is the case for example in glomerulonephritis, an autoimmune disease.
14.2 Mechanism of renal glucose loss 251
Notes: The bulk of the metabolites, including glucose, and most of the water are
taken up again in the proximal tubule of the nephron. Other metabolites, such
as uric acid (slide 16.5.4), are secreted into the urine in the proximal tubule by
active transport. The distal segments of the tubule are concerned with fine-tuning
the urine volume, and with the secretion or retention of salt ions and protons
according to the currently prevailing metabolic situation.
14.2.6 The capacity for glucose reuptake is saturated slightly above the phys-
iological plasma concentration range
normal Reabsorption
range maximum Glucose
filtrated
Glucose filtrated or excreted
Glucose
excreted
Notes: The normal range of glucose in the blood is approximately 4–7 mM. Glu-
cose starts to appear in the urine when the plasma glucose level exceeds the
reabsorption maximum of ~10 mM. This level is easily exceeded in untreated or
inadequately treated diabetics, who are therefore prone to glucosuria. If glucose
is detected in the urine, this means that at some time during the last couple of
hours the blood glucose must have exceeded the reabsorption threshold. Testing
252 14 Diabetes mellitus
for glucose in the urine can thus be useful in assessing the stringency of glucose
control in a patient.
Due to its osmotic activity, glucose excreted in the urine will also cause an
increase in the urine volume; this effect is referred to as osmotic diuresis.2
Notes: Many metabolic pathways are regulated by the balance between glucagon
and epinephrine on the one hand and insulin on the other. The level of the intra-
cellular second messenger cyclic AMP (cAMP) is a key parameter that represents
this balance. Glucagon and epinephrine activate adenylate cyclase, which forms
cAMP, whereas insulin activates phosphodiesterase, which cleaves cAMP. If insulin
is lacking or insulin sensitivity is diminished, the level of cAMP will go up, and
protein kinase A (PKA) will be activated.
As we have seen, one of the target proteins controlled by cAMP and PKA
is the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase.
Phosphorylation of this enzyme activates the bisphosphatase, which lowers the
level of fructose-2,6-bisphosphate (see slide 7.5.4). This inhibits glycolysis and
activates gluconeogenesis (see slide 7.5.3).
Excessive cAMP levels also affect glycogen metabolism, such that glycogen
synthesis is inhibited and breakdown is increased (see slide 8.4.2). Therefore,
even though blood glucose is already high to due to lack of cellular uptake in
peripheral tissues, both gluconeogenesis and glycogen breakdown in the liver are
activated, producing more glucose and further compounding the excessive glucose
accumulation.
14.1 How does diabetes affect the rate of gluconeogenesis?
2
Osmotic diuresis can also be induced with other solutes. The sugar alcohol mannitol has been
used in this manner to speed up the elimination of drugs and poisons, but this treatment appears
to be no longer in common use.
14.3 Metabolic dysregulation in diabetes mellitus 253
protein kinase A
hormone-sensitive lipase
fatty acids,
triacylglycerol
glycerol
Notes: The cAMP/PKA cascade also activates the hormone-sensitive lipase in fat
tissue. This enzyme initiates the breakdown of triacylglycerol to free fatty acids
and glycerol, which are then released into the blood stream. Glycerol will feed into
gluconeogenesis in the liver. Fatty acids may be directly consumed or feed into
ketogenesis in the liver.
protein
TCA
cycle
amino acids keto acids
malate
pyruvate glucose
pyruvate glutamate
glutamate
α-ketoglutarate
α-ketoglutarate
alanine alanine
Notes: In skeletal muscle, the lack of insulin prevents uptake of glucose, which
creates a need for alternative sources of energy. In part, this need is filled by fatty
acids and ketone bodies, but additionally protein degradation is activated.3
The amino acid released by protein breakdown also enter degradation. As we
have seen, many amino acids are converted to pyruvate or to TCA cycle interme-
diates (slide 12.1.2); the latter can also be converted to pyruvate by malic enzyme
3
Indeed, protein degradation is activated beyond the muscle’s own metabolic need by signaling
pathways that also cause muscle wasting in chronic inflammation and cancer [79].
254 14 Diabetes mellitus
(see slide 9.3.2). Pyruvate is transaminated to alanine, which travels to the liver
and enters gluconeogenesis. Alanine formation and transport to the liver also
occur as part of the glucose-alanine cycle (slide 12.3.5). The difference between
the regular function of that cycle and the catabolic situation in untreated diabetes
is that glucose cannot reenter the muscle cells; this greatly accelerates the loss of
substrate carbon from muscle.
14.2 What happens to protein metabolism in muscle in diabetes?
cholesterol lipoproteins
Notes: Ketone bodies are fairly strong acids. The kidneys normally compensate for
the accumulation of ketone bodies in the blood through the secretion of protons
in the form of ammonium chloride. However, the excessive rate of ketogenesis
14.3 Metabolic dysregulation in diabetes mellitus 255
in acute diabetes tends to overwhelm the capacity of the kidneys to balance the
blood pH. The condition is referred to as ketoacidosis. 4
Observation Cause
Symptom Cause
Notes: In addition to dehydration and pH deviation, one might consider high ace-
tone levels as an additional cause of diabetic coma, since acetone has documented
narcotic activity [80, 81]. This possibility has been considered in a study on sev-
eral patients that had experienced diabetic coma [50]; the authors concluded that
plasma acetone levels in these patients fell short of narcotic concentrations.
14.4 How is acetone formed in diabetic metabolism, and what is its significance?
4
Other possible forms of acidosis are accumulation of lactate (lactic acidosis) and respiratory
acidosis, which is due to insufficient removal of CO2 through the lungs. Lactic acidosis can be
induced by oral metformin and related oral antidiabetic drugs (see slide 14.5.14).
256 14 Diabetes mellitus
Notes: Coxsackieviruses belong to the enterovirus group, like hepatitis A virus and
poliovirus. These viruses contain a single-stranded RNA genome and are “naked”,
that is, they do not possess a lipid envelope.
Coxsackieviruses can cause non-specific febrile disease but occasionally give
rise to pleurodynia, which is characterized by chest pain during breathing that
results from the infection of muscles in the rib cage. These viruses may also cause
myocarditis, that is, an infection of the heart muscle. You will not be surprised to
hear that, of the two, myocarditis is the much more serious condition.
The involvement of coxsackieviruses in the pathogenesis of type 1 diabetes is
not due to direct damage to the pancreatic β-cells by the virus; instead, it arises
from an immunological cross-reaction between viral antigens with cellular antigens
found in the β-cells.
• T-helper cells,
• T-suppressor cells, and
• T-killer cells (cytotoxic T cells).
The interaction between lymphocytes and other nucleated cells that enables
the lymphocytes to recognize and eliminate virus-infected cells is controlled by
two protein molecules. These two proteins are the HLA molecules, which we find
on all nucleated cells, and the T cell receptors, which occur only on T lymphocytes.
HLA molecules present potentially antigenic peptides to the T cell recep-
tors. These peptides are samples drawn randomly from the entire set of cellular
proteins: As proteins undergo turnover and degradation within the cell, some
of the ensuing peptide fragments will become bound to HLA molecules and be
transported to the cell surface.5 If the cell is not infected by a virus, these surface-
exposed peptides will all be fragments of human-encoded proteins; on the other
hand, if the cell is virus-infected, a lot of these peptides will be of viral origin.
The HLA-associated peptides are recognized by T cell receptors. These recep-
tors occur in very many variants that differ in antigen specificity. The variants are
produced through combinatorial rearrangement of DNA fragments in the corre-
sponding genes of the T cells, which means that all receptor molecules found on a
given T cell, as well as on all of its daughter cells, will be identical.6
Since a T cell receptor arises through a randomized DNA recombination pro-
cess, its antigen specificity is also random; it may have affinity for a self antigen,
that is, a macromolecule that occurs normally in the body and has every right
to be there, or for a non-self antigen such as viral protein. Any T lymphocytes
that recognize self antigens are normally weeded out by the immune system it-
self; this is the role of the T suppressor cells.7 In contrast, T helper cells and
T killer cells that have no auto-reactive specificity are allowed to persist, since they
may have useful affinity for non-self antigens such as viral proteins. Once such
a T cell comes across another nucleated cell that happens to present its cognate
antigenic peptide, it will bind to this cell and attack it. The T cell-mediated killing
of virus-infected cells is a key process in antiviral immune defense.
The random generation of T cell clones with novel antigen specificities may
also produce cross-reactive clones that recognize both self antigens and viral
antigens. Such clones will usually be suppressed, but occasionally an acute virus
infection may intercede and cause them to proliferate before the suppression takes
hold. This may then result in damage to cells that express the cross-reacting self
antigen. Such a cross-reaction may occur between coxsackieviruses and some self
5
The abbreviation HLA stands for human leukocyte antigen, but HLA molecules are found on all
nucleated cells, not just leukocytes. They are, however, not found on red blood cells, which have
no protein synthesis and thus no use for HLA molecules. HLA molecules are very diverse between
individuals; this is what makes them antigens. Their absence on red blood cells explains why blood
transfusions are far less fraught with immunological incompatibilities than are organ transplants.
6
A similar mechanism also produces the clonal variation of antibody specificities in B lympho-
cytes.
7
Failure of the T suppressor cells to eradicate autoreactive T helper or T killer cells will result in
autoimmune diseases.
258 14 Diabetes mellitus
antigens of pancreatic β-cells, causing the destruction of β-cells and giving rise to
type 1 diabetes.
T cell receptor
HLA residues that
interact with the
T cell receptor
presented peptide
HLA molecule
Notes: The alleles and associated risks listed here are just one illustrative example
of the influence of HLA genotypes on the risk of contracting type 1 diabetes. The
relative risks are calculated to the average risk of the population; the absolute risks
represent the probabilities of contracting type 1 diabetes for persons carrying the
allele in question. Note that 94% of the individuals with the high-risk allele will not
contract diabetes.
8
The diversity of HLA antigens among humans is believed to have arisen through the exposure
to different infective agents that selected for different HLA antigens. Association of specific HLA
alleles with protection from infection is documented for various pathogens, including HIV [82].
14.5 Therapy of diabetes 259
Notes: New, untreated cases of diabetes present themselves with different degrees
of clinical urgency. The acute illness and coma scenario is more likely to occur in
diabetes type 1 than type 2, since in the former insulin secretion subsides acutely,
whereas in the latter things tend to go downhill more slowly. Acutely ill patients
will require both phases of treatment outlined in this slide, whereas others will
seek treatment at less advanced stages and can enter the second stage of treatment
right away.
Acutely sick diabetes patients typically have lost a lot of fluid and salts. They
are likely in acidosis, that is, their blood pH is too low due to the accumulation of
ketone bodies. All these deviations must be corrected by infusion therapy. Insulin
is initially administered with the infusion. All components of the infusion, and the
flow rate of the infusion itself, are adjusted according to frequent laboratory tests.
When the acute situation has been brought under control, the infusion therapy
is terminated and the patient is returned to oral nutrition. The next task is to
find out how much insulin this individual patient needs to match their appropri-
ate caloric intake. This usually takes a few weeks of experimentation. Once an
appropriate schedule for diet and insulin injections has been worked out and the
patient has been properly trained, they can be discharged, which is followed by
initially frequent ambulant supervision.
14.6 Which problems need to be addressed in the treatment of an acute case of type 1
diabetes?
Notes: The blood level of insulin rises up rapidly immediately after a meal and
then slowly declines over a time course of several hours. The slow pace of the
decline is due to sustained secretion of insulin at slowly decreasing rates; individ-
260 14 Diabetes mellitus
ual insulin molecules that have been secreted into the plasma decay much faster,
with a half-life of only ~15 minutes. This rapid inactivation is brought about by
peptidases in the blood plasma. If we simply inject insulin intravenously, it will
be just as rapidly degraded as the endogenously secreted hormone; in fact, this is
what happens to insulin that is applied with the infusion treatment in the acute
stage.
postprandial
peak
plasma insulin level
sustained
secretion
time
Continuous infusion is, of course, not practical for long-term treatment; instead,
we need a way to mimic the slow, protracted changes of the physiological insulin
profile with a limited number of injections per day. The following slides illustrate
how this is accomplished.
14.5.3 The reversible aggregation of insulin delays its diffusion from tissue
into the circulation
Hexamer Dimer Monomer
Capillary wall
Notes: When we inject the insulin into the tissue (typically into the subcutaneous
fat tissue—not into the cells, but rather into the fluid-filled interstitial space in
between) instead of intravenously, insulin has to diffuse across the walls of the
capillaries to reach the circulation, which will occur in a protracted fashion. At high
concentration, the extravascular insulin will form hexameric complexes, which are
too large to freely diffuse across capillary walls; the required dissociation into
dimers and then monomers will further delay the uptake.
The extent of insulin aggregation varies with the ion composition and pH in the
preparation. For example, zinc ions stabilize the hexamer, and a low pH induces
14.5 Therapy of diabetes 261
aggregation of hexamers into larger complexes, from which the insulin will take
even longer to fully dissociate. These effects can be used to tweak the intended
rate of uptake in clinically used insulin preparations.
protamine MARYRCCRSQSRSRYYRQRQRSRRRRRRSCQTRRRAMRCCRPRYRPRCRRH
Notes: Another effective trick for tweaking the rate of release of insulin is to
complex it with protamine, a small, basic DNA-binding protein that occurs in
sperm cells. Insulin itself is negatively charged at neutral pH; the two proteins
simply cluster together due to ionic interaction.
While protamine complexes and other measures to promote insulin aggrega-
tion cause delayed and protracted uptake of insulin into the circulation, another
useful technique is to derivatize insulin so as to increase its stability in the circu-
lation. A derivative that has been christened “insulin detemir” carries a fatty acyl
residue, which causes it to reversibly bind to albumin; in the albumin-bound state,
it is protected from degradation by circulating peptidases.
14.7 How does insulin aggregation affect the rate of systemic uptake of subcutaneously
injected insulin?
short-acting
plasma insulin
long-acting
combination
time
Notes: As pointed out above, insulin preparations can be tweaked and tuned
for a faster or slower time course of action. In order to mimic both the steep
increase and the slow decline of the physiological insulin secretion profile (see
slide 14.5.2), fast-release and slow-release preparations are mixed into so-called
biphasic insulins.
For many decades, biphasic and slow-release insulin preparations, applied two
to three times per day, were most widely used in insulin substitution therapy.
262 14 Diabetes mellitus
However, the accuracy of glucose control that can be achieved with such regimens
is limited, and the current trend favors more frequent insulin injections for tighter
glucose control. While this is less convenient for the patient, it does help to avoid
or postpone long-term complications of diabetes (see below).
Deviation Symptoms
Notes: Insulin substitution treatment can go off the rails when the endocrine and
metabolic situation of the patient changes, while the insulin dosage does not. For
example, acute infections may drive up cortisol secretion, which in turn will raise
blood glucose; on the other hand, loss of appetite, vomiting or diarrhea will reduce
glucose uptake and blood levels.
In extreme cases, both low glucose levels (hypoglycemia) and excessively high
ones (hyperglycemia) can induce coma. Among the two forms, hypoglycemic
coma is more common and more immediately life-threatening. Therefore, in case
we should find a comatose patient with known diabetes, and we are unable to
determine whether he is hyper- or hypoglycemic—a largely hypothetical case now
that glucose meters have become ubiquitous—we would inject glucose first. If the
patient wakes up, the patient was indeed hypoglycemic; if not, we would assume
the opposite and would then try insulin.
[83]. Elevated blood lipids (slide 14.3.4), protein glycosylation (slide 11.6.7), and
lack of C-peptide [76] are other potential mechanisms of long-term tissue damage.
Even though the biochemical mechanisms that underly the pathogenesis of
diabetic long term complications are not fully understood, it has been observed
that the severity of the clinical picture correlates with the cumulative extent and
duration of upward deviations in the blood glucose level.
In the early days of insulin substitution therapy, the blood glucose used to
be adjusted to levels somewhat above the normal range; this was done in order
to prevent hypoglycemia, which could be acutely life-threatening. However, while
sufficient to prevent acute symptoms, this approach favors the occurrence of long-
term complications. Intensive insulin therapy (slide 14.5.9) addresses this problem.
H2 O
delayed reaction
immediate reaction
Signal
Time
Notes: In regular physiology, the blood levels of glucose are connected by a neg-
ative feedback loop: As glucose rises, insulin rises and causes glucose to drop;
conversely, a dropping glucose level will reduce the output of insulin, which then
causes the glucose level to rise again.
Negative feedback loops operate in many places in metabolic regulation and
ensure the homeostasis of metabolite levels. To be effective, the response of a
negative feedback loop to changed input must be swift, since delayed feedback
will cause overshooting corrections and induce oscillations.
In intensive insulin therapy, the ideal goal is to replace the function of the
physiological glucose-insulin feedback loop. Apart from timely injections of in-
sulin, this also requires that the injected insulin quickly reaches the bloodstream
and becomes active. If the insulin effect takes hold too slowly after injection, this
will cause delayed feedback and potentially dangerous oscillations of the glucose
level.
Notes: Several insulin mutants optimized for rapid uptake contain substitutions
of the proline residue in the 28th position of the B chain. The effects of these
mutations can be understood from the structure of insulin aggregates.
14.5 Therapy of diabetes 265
Dimer 1
Gly B23
Glu B21
Pro B28
Dimer 2 Dimer 3
The structure on the left is that of an insulin hexamer, consisting of three dimers.
In each dimer, one monomer is shown in yellow and the other in blue; the green
balls in the center are zinc ions. On the right, it is shown how the hydrophobic pro-
line residue in position B28 of one monomer interacts favorably with the adjacent
side chains of the second monomer; this interaction stabilizes the dimer.
Substitution of proline with a charged residue breaks this favorable interaction.
In insulin aspart, the proline is replaced with aspartate, which creates electrostatic
repulsion with glutamate B21 on the other subunit. In insulin lispro, proline
B28 and lysine B29 are swapped, which also reduces the mutual affinity of the
adjoining monomers. Mutants like these are currently widely used in intensive
insulin therapy.
14.9 What is the purpose of intensive insulin therapy? What are the tools?
Rosiglitazone O Tolbutamide
O
H
NH S N
N N S H
O O N
O O
HO N O O O OH
NH2 NH
OH H OH OH OH OH
Acarbose Metformin
Notes: Oral antidiabetic drugs are used only in type 2 diabetes. These structures
are just for illustration—no points for memorizing them.
266 14 Diabetes mellitus
Notes: The action mode of tolbutamide was covered earlier (slide 13.2.8). Since
its role is to stimulate insulin secretion from pancreatic β-cells, it is clear that it is
useless in type 1 diabetes, since most or all β-cells are destroyed in this case.
Rosiglitazone is an agonist of the PPARγ, a nuclear hormone receptor that
regulates the transcription of numerous enzymes and transporters involved in
glucose and fat metabolism. It reduces insulin resistance in type 2 diabetes, but
it has numerous side effects including the promotion of heart failure that have
led to its withdrawal from the market in Europe. Efforts to find similar drugs with
fewer side effects are ongoing.
Aldose reductase inhibitors have been considered briefly above (slide 14.5.7),
and metformin will be considered below.
NAD+ Lactate
AMP-activated
4
protein kinase
AMP ATP
NADH Pyruvate
3
1
Metformin
1
2 e– 2 O2 ADP ADP
NAD+ 2 H+ Pi
H+ H2 O H+ ATP
Notes: Metformin and related biguanide drugs are probably the most widely used
oral antidiabetics. In vitro, metformin has been shown to inhibit NADH dehydroge-
nase (1), that is, complex I of the respiratory chain. As a consequence, the activity
of ATP synthase (2) should drop, too. This would increase the level of ADP, and
adenylate kinase (3) should cause AMP to rise as well.
14.6 Answers to practice questions 267
The regulatory effects of AMP resemble some of those brought about by in-
sulin (slides 7.5.3f). In addition, there is an AMP-activated protein kinase that
participates in metabolic regulation. Among other effects, this regulatory enzyme
activates insulin-independent glucose uptake by cells that normally do require
insulin. Another potentially relevant effect of AMP is the inhibition of adenylate
cyclase [84], which would counteract the effect of glucagon and epinephrine, and
so help to restore the balance between insulin and its antagonists.
If this proposed mechanism is correct, metformin should also cause accumu-
lation of NADH. This would drive lactate dehydrogenase (4) into reverse mode,
producing lactate. Lactate acidosis is indeed a known complication of metformin
therapy.
125
respiration rate (% of control)
100
24 hours
60 hours
75
50
50µM 100µM 50µM 100µM metformin
Notes: This slide (data from [85]) shows some experiments supporting the mech-
anism depicted in the preceding slide. Metformin inhibits the respiratory activity
of mitochondria when fed with malate but not when fed with succinate. Malate
dehydrogenase produces NADH, which delivers electrons to complex I. In contrast,
succinate dehydrogenase produces FADH2 , which feeds its electrons to complex II
and so bypasses the blockade of complex I.
Various aspects of the action mode presented here have been called into ques-
tion by several experimental studies. One such study attempted but failed to ob-
serve the expected concomitant change in the [ADP]/[ATP] ratio [86]. Furthermore,
genetic knockout of AMP-activated kinase does not abolish the glucose-lower-
ing action of metformin [87]. Therefore, despite the long-standing clinical use
of biguanide drugs, their mode of action is not yet fully understood.
14.10 What is the mode of action metformin?
15.1 Overview
The transfer of single carbon units, in various redox states, is important in several
biosynthetic pathways. The two coenzymes tetrahydrofolate and methylcobalamin
play central roles in these reactions. Both coenzymes are derived from vitamins,
and deficiencies of these vitamins are not uncommonly encountered in clinical
medicine, usually as a consequence of another underlying medical condition.
We will first look at the biochemical reaction cycles and then consider the
pathological consequences that result from their disruption through vitamin defi-
ciencies.
Notes: The degradation pathways for several amino acids produce single carbon
units that are transferred to tetrahydrofolate (THF) as an intermediate carrier.
269
270 15 Biosynthetic pathways using tetrahydrofolate and vitamin B12
O OH
p-aminobenzoate O polyglutamate
N OH
H
O
OH HN n
pteridine moiety N
N
H2 N N N
NADPH+H+ NADPH+H+
H2 N N N H2 N N N H2 N N N
H H
Notes: Folic acid is the vitamin as it is found in the diet. It consists of three
distinct moieties: a pteridine moiety, a p-aminobenzoate moiety, and one or several
glutamate residues.
The metabolically active form is tetrahydrofolate, which is formed from fo-
late in two successive NADPH-dependent reductions, both catalyzed by the same
enzyme, namely, dihydrofolate reductase.
The number of glutamate residues contained in folic acid or tetrahydrofolate
(THF) changes at various stages of transport and utilization. Intestinal uptake
and transport through the blood occur with only one glutamate attached. After
uptake into liver cells, several more glutamate residues are added. The number
of glutamate residues present is involved in the regulation of folate-dependent
metabolism.
Sources:
1. serine, glycine
2. histidine, tryptophan
Biosynthetic destinations:
1. purine bases
2. thymine
3. S-adenosylmethionine choline phospholipids, creatine, epinephrine, DNA
methylation
15.2 Tetrahydrofolate-mediated carbon transfer reactions 271
Notes: Degradation of the amino acids glycine, serine, histidine and tryptophan
provides C1 units to tetrahydrofolate. Serine and glycine are more abundant than
the two others and therefore are the main sources.
15.1 Summarize the role of tetrahydrofolic acid in metabolism.
O N
CH H ⊕ CH
H2 O
⊖O ⊖O
P P
⊕ ⊕
N N
Enzyme H Enzyme H
B ⊕B H
H H
O CH2 H C COOH O CH2 C COOH
N N
H ⊕ CH H ⊕ CH
⊖O ⊖O
P P
⊕
N N
H H
Notes: One of the major reactions that supply C1 units to THF is the cleavage
of serine by serine hydroxymethyltransferase. The active site of this enzyme
contains the coenzyme pyridoxal phosphate (PLP) and a catalytic base (here
represented by B). As in amino acid transamination (section 12.2), the α-amino
group of the substrate serine forms a Schiff base with PLP. Through this bond,
PLP can reversibly withdraw and donate electrons, which in cooperation with the
catalytic base facilitates cleavage of the bond between the α- and the β-carbon. The
β-carbon is released as formaldehyde, and the aldimine is subsequently hydrolysed
to release glycine (not shown).
Note that the reaction is reversible and therefore can bring about the synthesis
of serine from glycine and N,N ′-methylene-THF.
NH3 NH3
R R
HO HN HO H2 C N
H
N N
N N
H2 O
H2 N N N H2 N N N
H H
S
O
NADH + H+ H2 N
OH
O
NH
+
NAD CO2
O
HN N
H
SH O
SH
HS
S
H2 N
NH3 + N,N’-CH2 -THF THF
imidazolone- formimino-
histidine urocanate propionate glutamate
HO HO H2 O HO H2 O HO
O O O O
H2 N
O O
NH3
N NH N NH N NH HO NH
NH
HO NR NH THF
⊕N H+ HO NHR
N N
N
H2 N N N
H H2 N N N
NH3 H glutamate
N,N′ -methenyl-THF
+
NADPH NADP
H2 O
N N N
H H H
CO2
NADPH
NADP+
R R
CH3 N NADH H2 C N
N N
NAD+
N N
H H
Notes: C1 derivatives of THF exist in different redox states that can be converted
into one another. The final reduction, which leads from N,N ′-methylenetetrahy-
drofolate to N 5 -methyl-THF, is apparently irreversible; this is relevant for the
methyl trap hypothesis that accounts for the pathogenesis of anemia in vitamin
B12 deficiency (see slide 15.5.6).
274 15 Biosynthetic pathways using tetrahydrofolate and vitamin B12
B12
O CH3
S
H CH3
N HN
N
CH
HC
N N O N OH
H H2 N
O
Notes: Folic acid metabolism in microbes contains drug targets for antimicrobial
therapy. An important example are the sulfonamides, which became available
in the 1930s and were the first broad-spectrum antibacterial drugs. The first
such drug, sulfamidochrysoidine (trade name Prontosil), was developed as a red
dyestuff. Its antibacterial activity was discovered in a test program that systemati-
cally evaluated the effects of all the company’s new compounds in animal experi-
ments.
The compound as such, when tested in vitro, has no antibacterial activity.
The active component is sulfanilamide, which is released in animals or humans
15.4 Cobalamin-dependent methylation reactions 275
O Cl
N S NH2
H2 N N O N
NH2 H2 N N NH2
Sulfanilamide O Trimethoprim
H2 N S NH2 O
O O O
p-Aminobenzoic acid O N
H2 N
OH H2 N N NH2
Notes: Cobalamin is a coenzyme with a complex structure. Its corrin ring coordi-
nates a single cobalt ion, which is central to the ability of cobalamin to facilitate
the cleavage and formation of carbon bonds by way of radical intermediates.
276 15 Biosynthetic pathways using tetrahydrofolate and vitamin B12
O NH2
H2 N
O
H2 N
NH2
O
N N O
Co2+ O
N N⊖ NH2
O
H2 N O NH
AM P
ATPi 3 Pi
THF−CH3 B12
precursor
methylated product
THF B12 −CH3
adenosine H2 O
N N
N N
S⊕ S
H3 C N N N N
O O
OH OH OH OH
SAM SAH
Notes: The key feature of SAM is the methyl group attached to a sulfonium ion
(S+ ). The inherent instability of the sulfonium group makes this compound a good
methyl group donor.
S-adenosylhomocysteine (SAH) is not only the byproduct of SAM-dependent
methylation reactions but also a competitive inhibitor. It must therefore be re-
cycled promptly in order to avoid disruption of the SAM cycle. In vitamin B12
deficiency, the regeneration of SAM from SAH is impeded, and the accumulating
SAH interferes with subsequent methylation reactions [89].
⊕
NH2 NH N N
O O O O
O O O O
P P P P
O− O− O− O−
O O O O
O O O O
O O O O
O O O O
O O O O
PE methyl-PE dimethyl-PE PC
N+ N+
O O
O O
P P
O− O−
O HO HO O
H H
O N O N
O O OH O O OH
O O
O O
15.7 What is the SAM cycle? At what point in the cycle is vitamin B12 required?
Notes: Myelin sheaths consist of multiple layers of cell membranes, which contain
more phospholipids and less protein than most other cell membranes. These
elaborate structures enable the saltatory conduction of action potentials, which
is much faster than the pedestrian non-saltatory conduction that occurs along
non-myelinated fibers. The picture shows a cross section through a single nerve
15.4 Cobalamin-dependent methylation reactions 279
fiber; the thick, dark zone surrounding the axon is the myelin, which consists of
multiple stacked membrane bilayers.1
Cobalamin deficiency causes demyelination or nerve fibers in the central and pe-
ripheral nervous system. The neurological, and in advanced stages neuropsy-
chiatric, consequences can be severe. In addition to the disruption of PC and
sphingomyelin biosynthesis, deficient methylation of myelin-associated proteins
has been considered as another pathogenic mechanism [89].
NH+
2 NH+
2
ATP
creatine kinase
ADP
NH+
2
HN C NH P
NH N CH3
N CH2
H3 C O
P
COO−
Notes: The first two steps in creatine synthesis occur in the kidney and the liver,
respectively; the second step involves S-adenosylmethionine. Most of the creatine
1
The number of stacked bilayers surrounding the axon in this picture is approximately 20, but it
can be substantially higher in the fastest-conducting axons.
280 15 Biosynthetic pathways using tetrahydrofolate and vitamin B12
Since both folate and B12 are vitamins, deficiency arises from disruptions of intesti-
nal uptake. We therefore need to understand how this intestinal uptake occurs.
Notes: In macrocytic anemia, the number of blood cells is reduced, but the size of
the individual cells is increased. With red blood cells, the content of hemoglobin
per cell is also increased. On the whole, however, the hemoglobin content of the
blood is decreased, which makes this condition a form of anemia.
C1 units derived from THF are required at several points in nucleotide biosyn-
thesis (see slide 15.2.9). The inhibition of nucleotide biosynthesis that results from
the lack of THF will interfere with DNA synthesis and cell division more strongly
than with protein synthesis. Protein synthesis “outruns” DNA synthesis, leading
to the accumulation of more protein per cell between successive cell divisions. In
the precursor cells of erythrocytes, this causes a greater than normal hemoglobin
content per cell.
282 15 Biosynthetic pathways using tetrahydrofolate and vitamin B12
15.5.4 The intestinal uptake of vitamin B12 involves multiple carrier proteins
Notes: The intestinal uptake of vitamin B12 is a rather intricate affair. The vitamin
contained in the food is bound to enzymes or other proteins. In the stomach, these
are denatured by gastric acid and cleaved by pepsin. The vitamin thus released is
immediately bound by the carrier protein haptocorrin, which is secreted by the
gastric mucous membrane.
When the stomach contents reach the small intestine, haptocorrin itself is
degraded by trypsin or other pancreatic proteases, and vitamin B12 is released
again. The free vitamin is then recaptured by intrinsic factor, a second carrier
protein that is also produced by the gastric mucosa (and which up to this point
simply came along for the ride). The B12 -intrinsic factor complex then travels all
the way down to the terminal ileum, the lowermost section of the small intestine,
where it is taken up by receptor-mediated endocytosis. Inside the cells of the gut
epithelium, vitamin B12 changes carriers for a third time; bound to its final carrier,
transcobalamin, the vitamin then reaches the liver and all other tissues beyond.
The daily uptake of vitamin B12 is on the order of only 1-5 µg. The liver stores
about 2-5 mg. When uptake is disrupted, this store will last a long while before
vitamin deficiency becomes clinically manifest.
15.5 Folate and vitamin B12 deficiency syndromes 283
histidine serine
B12
formyl-THF methionine
Notes: Like folic acid deficiency, a lack of vitamin B12 causes macrocytic anemia,
and the changes to the cells found in the peripheral blood and in the bone marrow
are indistinguishable between the two conditions. In folate deficiency, the macro-
cytosis can be explained by the inhibition of nucleotide synthesis (see slide 15.5.3).
However, unlike folic acid, cobalamin itself is not required for those synthetic path-
ways (see slide 15.2.9). How, then, can we account for the clinical resemblance?
This is explained by the methyl trap hypothesis.2
When vitamin B12 is deficient, S-adenosylmethionine (SAM) will be lacking (see
slide 15.4.2). SAM imposes feedback inhibition on the reduction of N,N ′-methy-
lenetetrahydrofolate to methyl-THF; therefore, the lack of SAM permits excessive
accumulation of methyl-THF. The high level of methyl-THF, in turn, inhibits serine
hydroxymethyltransferase, which is one of the major reactions that supply C1
units to the THF pool (see slide 15.2.4). The C1 -THF pool becomes depleted. In
keeping with this explanation, folate substitution does transiently improve the
blood count in patients with B12 deficiency.
A lacking capacity for biosynthetic methylation also interferes with the syn-
thesis of phospholipids, which in turn causes demyelination of nerve fibers (see
section 15.4). The neurological consequences tend to be more grave in vitamin B12
deficiency than in folate deficiency.
15.8 Explain why vitamin B12 deficiency causes macrocytic anemia.
2
Some theoretical principles seem to be forever stuck with the byword “hypothesis,” such as
the current one or the “Mary Lyon hypothesis” (see slide 9.4). Both hypotheses are actually well
supported by observation; see [96] for experimental evidence supporting the methyl trap hypothesis.
15.6 Answers to practice questions 285
15.3: The glycine cleavage system is a multi-enzyme complex that consists of three dif-
ferent enzymes and a substrate carrier protein with a lipoamide moiety. I cleaves glycine
according to the following equation:
Glycine + NAD+ + THF NH3 + CO2 + NADH+H+ + N,N′ -methylene-THF
15.4: The C1 carbon can be carried by THF as a formyl, methenyl, methylene, or methyl
group. Purine synthesis uses formyl-THF, thymidylate synthase uses methylene-THF, and
methionine synthesis uses methyl-THF.
15.5: Folic acid is synthesized by microbes but not by human cells. Therefore, inhibitors
of folate synthesis are selectively toxic for microbes and can be used for antimicrobial
therapy. Sulfonamides are the predominant class of folate synthesis inhibitors.
Dihydrofolate reductase occurs in both microbial and human cells. Inhibitors that se-
lectively target the microbial enzymes are available and are used for antimicrobial therapy,
preferably in conjunction with sulfonamides. Inhibitors of the human enzyme are used in
cancer therapy and for immunosuppression.
15.6: See slide 15.4.4.
15.7: The S-adenosylmethionine or SAM cycle is a pathway that mediates the transfer
of methyl groups from N,N ′-methyl-THF to phosphatidylethanolamine (PE) and various
other substrates. The first reaction, which is vitamin B12 -dependent, is the transfer of the
methyl group from methyl-THF to homocysteine, which yields methionine. Using ATP,
methionine is then activated to S-adenosylmethionine, which donates the methyl group to
various acceptors, including PE, epinephrine, and (cytosine bases in) DNA. The cleavage of
S-adenosylhomocysteine to adenosine and homocysteine completes the cycle.
15.8: In vitamin B12 deficiency, macrocytic anemia arises through the formation of exces-
sive amounts of methyl-tetrahydrofolate, at the expense of formyl- and methylene-THF,
which are required in the synthesis of nucleotides.
Vitamin B12 is required in the conversion of homocysteine to methionine, which is
then converted to S-adenosylmethionine. The latter exerts feedback inhibition on the
reduction of methylene-THF to methyl-THF. Lack of vitamin B12 disrupts this negative
feedback.
Chapter 16
Nucleotide metabolism
16.1 Introduction
Notes: Nucleotides also occur as parts of more complex cosubstrates and coen-
zymes, three of which are shown here. These three molecules have very different
“business ends.” In coenzyme A, the business end is the thiol group that becomes
bound to the substrate, and in NAD+ it is the nicotinamide moiety that under-
goes reversible reduction and oxidation. In 3′-phosphoadenosine-5′-phosphosul-
fate (PAPS), the key feature is the mixed phosphosulfate anhydride that activates
the sulfate toward transfer, in much the same way as the terminal phosphate is
287
288 16 Nucleotide metabolism
activated in ATP. In methylcobalamin, the chemically active center is the cobalt ion
that facilitates carbon bond cleavage and formation (see section 15.4).
O O coenzyme A NAD+
O
HS N N O
H H NH2
OH
−O P O ⊕
O N
NH2 O
PAPS O
NH2 −O P O N N
N N
−O P O OH OH
O− O− O O N N NH2
O
O S O P O O N N −O P O N N
OH O
OH OH
O O N N
O OH
−O P O
O− OH OH
The adenosine moiety found in each of these molecules, shown in blue, does not
directly participate in any of the catalytic functions; instead, it just serves as a tag
to facilitate recognition of the cosubstrates by the corresponding enzymes. Why,
then, do cosubstrates so often possess nucleotides as their binding tags, rather
than for example amino acids or peptides? A plausible answer is provided by the
RNA world hypothesis.
Notes: The RNA world is a hypothetical early stage of evolution. In this early world,
RNA was the predominant macromolecule. RNA not only stored and propagated
genetic information, a role that is almost universally filled by DNA in current life
forms, but it also realized and expressed this information, which in current life
forms is mostly accomplished by proteins.
The empirical basis of the RNA world hypothesis is that RNA can indeed
assume the role of DNA, as it still does in RNA viruses and viroids, and that it can
also have catalytic activity, as is the case in ribosomes and smaller ribozymes.
16.1 Introduction 289
The structure of the ribosome also suggests an evolutionary path along which
RNA enzymes may have been replaced by protein enzymes in evolution. The
catalytic centers of a ribosome (left) consist entirely of RNA (blue). The tiny red
dot in the center represents the antibiotic chloramphenicol, lodged in one of the
active sites of a bacterial ribosome; we are peeping at it through the ribosome’s
peptide exit tunnel. Ribosomal proteins (green) serve in structural and auxiliary
roles.
Like ribosomes, other RNA enzymes may at first have coopted peptides as
structural components, and possibly as coenzymes. The peptides may then have
grown into ever more important roles within the hybrid molecules, until they took
over entirely and made the RNA component obsolete.
16.1.4 Why have cosubstrates become fossilized, whereas enzymes have not?
... Me enzyme 4
Me substrate 1
... ...
... ...
substrate 2 ...
Notes: In a world dominated by RNA, one would expect cosubstrates and carrier
molecules to contain some nucleotide moieties also. However, if the above scenario
for the evolutionary replacement of RNA enzymes by protein enzymes is correct,
why did a similar replacement not occur with cosubstrates?
Let us consider the world from the perspective of an enzyme and of a cosub-
strate, respectively. An enzyme’s world is simple, containing one or a few sub-
strates and maybe a cosubstrate or coenzyme. If we make some random change
to the structure of the enzyme, there is a reasonable chance that the interactions
with all of these ligands will remain intact.
In contrast, a cosubstrate such as ATP or NADH inhabits a much more complex
world, containing a very large number of interacting enzymes. Chances are that a
change to its molecular structure would disrupt its interaction with some enzyme
molecule or other that is essential to the survival of the cell. Structural changes
to cosubstrates are therefore “forbidden,” and they may well look now exactly as
they did shortly after life first arose billions of years ago.1
1
The acyl carrier protein (ACP) domain that is found in fatty acid synthase is basically coenzyme
A with adenosine replaced by a peptide moiety (see slide 10.5.3). This is possible since ACP is private
property of fatty acid synthase and does not need to function with other enzymes.
The constraining effect of a large number of interactions can also be seen with peptides like
glutathione or proteins like calmodulin, which are also very strongly conserved throughout evolution.
290 16 Nucleotide metabolism
16.1 Explain the RNA world hypothesis, and the concept that nucleotide-based coenzymes
may be molecular fossils from the RNA world.
• De novo synthesis
• Intestinal uptake of nucleosides
• Endogenous turnover (partial degradation/salvage)
• Degradation and excretion
Notes: Synthesis of nucleotides from scratch occurs in all tissues, and its capacity
is sufficient to fully cover the needs of the organism; we do not require any nucle-
otides or bases in the diet. Nevertheless, nucleic acids contained in the diet are
broken down to nucleosides, which are taken up with high efficiency and degraded
(see section 16.4).
The pathways for the degradation of endogenous nucleotides have some over-
lap with those for dietary ones. Intermediates of nucleotide degradation can also
enter salvage pathways and then be reverted to complete nucleotides. We will now
look at all these pathways in turn.
There are two major synthetic pathways, for purine and pyrimidine bases,
respectively, both of which diverge towards their ends to produce the different
variants. We will begin with the pathway for purine synthesis.
O−
−O P O
O
O O O
O P O P O−
OH OH
PRPP O− O−
ring synthesis
IMP OH OH
Notes: Adenine and guanine nucleotides are derived from a common precursor,
inosine monophosphate (IMP), which consists of ribose phosphate and a purine
16.3 Purine synthesis 291
ribose-5-phosphate PRPP
O− O−
ATP
−O P O −O P O
O O
O O O O
OH AMP O P O P O−
OH OH OH OH
O− O−
glutamine
glutamate + PPi
H2 N
glycine + ATP
O− O−
−O P O HN O −O P O NH2
O O
O ADP + Pi O
OH OH OH OH
CO2
O OH
O O O
asp + ATP
N N N
NH2 N OH
H
N NH2 N NH2 O OH N NH2
fumarate ADP + Pi
R R R
O O O
N10 -formyl-THF
N N N
NH2 NH2 NH
N NH2 N N O N N
THF H2 O
H
R R R
Most of the chemistry in this pathway seems quite straightforward. Where ATP or
GTP come into play, they are used to phosphorylate hydroxyl groups, which are
thus activated for substitution by ammonia (released from glutamine) or by the
amino group of aspartate.
The most remarkable reaction appears to be the direct carboxylation of 5-
aminoimidazole ribotide (AIR) by AIR carboxylase, which unlike e.g. pyruvate car-
boxylase requires neither ATP nor biotin. Indeed, AIR carboxylase uses CO2 di-
rectly instead of ATP-activated bicarbonate, which is used by the biotin-dependent
enzymes. This is made possible by its energetic coupling with the subsequent
reaction (see next slide).
Notes: AIR carboxylase is actually part of a bifunctional enzyme that also contains
the next enzyme activity in the pathway (SAICAR synthetase), and the carboxylated
product (CAIR) is directly channeled to the SAICAR synthetase active site. The
16.3 Purine synthesis 293
two active sites are located close to the entrance and the exit, respectively, of a
connecting tunnel [97].
product
substrate substrate
The equilibrium of the AIR carboxylase reaction probably does not favor the prod-
uct (CAIR). In contrast, the SAICAR synthetase is driven forward by ATP hydrolysis.
Thus, the tunnel that connects the two active sites would seem to function like a
“vacuum cleaner” that removes all the product from the AIR carboxylate site and
thereby pushes that reaction forward also. Structure rendered from 2h31.pdb.
IMP O
N NH
O− GTP + aspartate
−O P O O N N
GDP + Pi O
O
OH
OH OH adenylosuccinate
OH
synthetase HN
N O
O− N
NH3
−O P O O N N
AMP deaminase O
H2 O OH OH
adenylosuccinate
NH2
N N
O−
adenylosuccinate lyase
−O P O O N N
O
fumarate
AMP OH OH
Notes: The synthesis of AMP from IMP occurs in two steps. In the first step, an
aspartate is acquired to form adenylosuccinate. The subsequent elimination of
fumarate—or, strictly speaking, the elimination of AMP from fumarate—is similar
to the conversion of SAICAR to AICAR (slide 16.3.3). Indeed, both reactions are
carried out by the same enzyme, which is named adenylosuccinate lyase after the
reaction depicted here.
294 16 Nucleotide metabolism
N
NH NAD+ + H2 O
O−
−O P O N N
O NADH + H+
O
IMP dehydrogenase O
OH OH
N
NH
O−
NADP+ + NH3 −O P O N N O
O
H
GMP reductase O
NADPH OH OH XMP
O
N
NH
O−
−O P O N N NH2 glutamine + ATP + H2 O
O
O
glutamate + AMP + PPi GMP synthetase
GMP OH OH
of PRPP itself, as do ADP and GDP (not shown). These effects adjust the flow
through the common pathway up to IMP in keeping with the total need for purine
nucleotide synthesis.
Ribose-5-phosphate
PRPP
PRA
IMP
XMP Adenylosuccinate
GMP AMP
GDP ADP
GTP ATP
The balance between AMP and GMP synthesis is maintained by the crosswise
cosubstrate requirement that was discussed above (green lines). In addition, GMP
and AMP both inhibit the formation of their immediate precursors from IMP; this
inhibition is competitive.
16.2 Give an overview of the biosynthesis of purine nucleotides and its regulation.
Notes: Nucleic acids are of nutritive value mainly because of the sugars they con-
tain, namely, ribose and deoxyribose. In the small intestine, pancreatic nucleases
(DNAse and RNAse) break down nucleic acids to nucleotides, which are dephos-
phorylated by alkaline phosphatase to nucleosides; the latter are taken up by
sodium-coupled active transport. There are two intestinal transporters that prefer
purine and pyrimidine nucleosides, respectively, but have fairly broad and over-
lapping specificity. This loose specificity permits piggyback uptake of nucleoside
analogue drugs such as idoxuridine and 6-mercaptopurine riboside (see section
16.9).
After uptake, nucleosides mostly undergo degradation in the liver. Cleavage
by purine and pyrimidine nucleoside phosphorylases gives free bases and ribose-
or deoxyribose-1-phosphate. The sugar phosphates are converted to mainstream
degradative intermediates via short adapter pathways (see next slide).
296 16 Nucleotide metabolism
nucleic acids
H2 O
DNAse, RNAse
nucleotides
H2 O
phosphatase
Pi
nucleosides
Na+
intestinal uptake
nucleosides nucleotides
Pi
phosphorylase salvage
degradation
Both free bases and uncleaved nucleosides can in principle be reused toward
the synthesis of nucleotides. However, the salvage pathways that accomplish this
appear to mostly process endogenously synthesized bases, whereas those obtained
from the diet mostly undergo degradation and excretion [99, 100].
phosphopentomutase
O− O−
−O P O −O P O
O O
O O
OH OH
OH OH OH
The following pathways account for the degradation of endogenous purine nucle-
otides, as well as of the free bases that result from the phosphorolysis of dietary
ones.
1 2
AMP IMP XMP GMP
3
adenosine inosine xanthosine guanosine
5 4
adenine hypoxanthine xanthine guanine
5
deamination
oxidation uric acid
dephosphorylation
phosphorolysis
Notes: As shown in slide 16.5.1, the degradation of AMP can follow several alter-
native routes that perform dephosphorylation, phosphorolysis, deamination, and
oxidation reactions in different sequences. This figure depicts one of these alter-
native routes in detail. The depicted reactions are catalyzed by AMP deaminase
298 16 Nucleotide metabolism
N N N N O N N
NH3 H NADH H
Ribose-P Ribose-P Ribose-P
H2 O
3
phosphate
O O O
H NAD+ phosphate
N N N
HN 5 HN 4 HN
O
O N N O N N O N N
H H NADH+H+ H H Ribose-1-P H Ribose
N N O N N H2 N N N
H H H2 O 2 H H NH3 H H
xanthine O2 + H2 O
oxidase
H 2 O2
O
H
N
HN
O uric acid
O N N
H H
16.5 Purine degradation and salvage pathways 299
urate urate
? URAT1
organic acid organic acid
urate urate
ATP? MRP4
Notes: Most of the uric acid formed by purine degradation is eliminated via the
kidneys. Urate is subject primarily to glomerular filtration and tubular reuptake
(see slide 14.2.5), while tubular secretion (by an ABC transporter named MRP4) is
less important.
Reuptake of urate from the primary filtrate is mediated by the URAT1 exchange
transporter. Several drugs and metabolites that affect renal urate elimination
interact with this transporter [101]. Typically, when present in the lumen of the
tubule, such compounds will compete with urate for reuptake; this is the mode of
action of uricosuric drugs like benzbromarone or probenecid. On the other hand,
when present inside the epithelial cells, they may act as substrates for exchange
and therefore increase the rate of reuptake, as is the case with pyrazinoic acid (see
slide 16.6.5).
Notes: While uric acid is the terminal product of purine degradation in humans
and other apes, many other organisms, ranging from fungi to mammals, perform
several subsequent reactions that degrade uric acid further to allantoin, which is
then excreted. Allantoin is more water-soluble than uric acid and therefore avoids
the problems that can arise from the low solubility of uric acid in humans (see
slide 16.6.2).
The first step in urate degradation is carried out by urate oxidase (uricase).
Interestingly, humans actually possess an inactive copy of the urate oxidase gene,
which indicates that at some point during evolution this enzyme activity was
lost. Since the genetic enzyme defect spread throughout the entire population,
there likely was some selective advantage to it. Several hypotheses have been
proposed as to the nature of this advantage, ranging from antioxidant effects of
uric acid over protection from UV irradiation to a rise in blood pressure. While
300 16 Nucleotide metabolism
the correlation between uric acid blood levels and hypertension is worth noting
from a clinical point of view [102], the biological advantage of an increase in blood
pressure is not obvious to me; and even if it existed, it would seem implausible for
evolution to cripple a perfectly fine pathway when a plethora of other physiological
mechanisms for adjusting the blood pressure was already available.
O O OH
O2 +H2 O H2 O 2
H H
N N
HN HN
O O
O N N urate oxidase (uricase) O N N
H H H
uric acid H2 O
spontaneous hydroxyisourate
hydrolase
H
O N O OH
spontaneous H
O N
HO
HN N O
H
HN N
H2 N O
CO2 H2 N O 2-oxo-4-hydroxy-
4-carboxy-5-ureido-
allantoin OHCU decarboxylase imidazoline (OHCU)
While the role of urate oxidase has been known for a long time, the subsequent
two enzyme reactions shown in this slide have only recently been discovered [103].
The reason for their delayed discovery is that urate oxidase alone is sufficient for
the conversion of urate to allantoin, since the subsequent reactions also occur
spontaneously. The major difference is that the spontaneous decay produces
allantoin as a racemic mixture, whereas enzymatic formation gives rise to one
enantiomer only.
Notes: Nucleosides and free bases that arise during the degradation of nucleic
acids and nucleotides can be salvaged and reused. This scheme shows the reac-
tions involved in purine salvage, which produce AMP, GMP and IMP. The conversion
of the latter to AMP or GMP (blue arrows) involves the same reactions as in de novo
synthesis (see slides 16.3.6 and 16.3.7).
Enzymes: 1, adenosine kinase; 2, inosine kinase; 3, guanosine kinase; 4,
adenine phosphoribosyltransferase (APRT); 5, hypoxanthine-guanine phosphori-
bosyltransferase (HGPRT). The APRT and HGPRT reactions are analogous to the
orotate phosphoribosyltransferase reaction (see slide 16.7.1).
16.6 Diseases related to purine degradation 301
nucleoside kinases
adenylo- phosphoribosyltransferases
succinate shared with biosynthesis
degradation
1 2 3
Uric acid
16.6.2 Gout
tion like other purine nucleotides and as such will contribute to uric acid produc-
tion, which may occasionally trigger gout [108].
fructokinase aldolase B
fructose fructose-1- P glyceraldehyde
dihydroxyacetone- P
ATP ADP
glyceraldehyde-3- P
1,3-bis- P -
3- P -glycerate Pi
PG-kinase glycerate
Notes: Fructose has been linked to increased uric acid production both experi-
mentally [109] and statistically [110]. The mechanism is as follows: Fructokinase
produces fructose-1-phosphate more rapidly than it can be turned over by aldolase
B. This ties up phosphate, which is then no longer available for the regeneration of
ATP. This raises the level of ADP, which is disproportionated by adenylate kinase
to ATP and AMP, and the latter enters degradation via AMP deaminase (to IMP) or
5′-nucleotidase (to adenosine; see slide 16.5.1).
In keeping with the assumption that uric acid formation is promoted by limit-
ing aldolase B activity, heterozygous carriers of a deficient gene for this enzyme
experience a greater increase of urate synthesis in response to a fructose challenge
than individuals with two intact gene copies [111]. As noted before, a homozygous
defect of aldolase B causes hereditary fructose intolerance. In this condition, phos-
phate sequestration and ATP depletion reach a much greater extent, with much
more immediate and disastrous consequences for the liver cells (see slide 4.2.2).
O O OH
O OH
N
NH N S Br
N N O O
H O
O OH O NH2 O OH O OH
HO
N N N
N N N
O
salicylic acid pyrazinamide pyrazinoate 5-hydroxy-
pyrazinoate
Some drugs or drug metabolites promote the tubular reuptake of uric acid, presum-
ably by functioning as exchange substrates at the URAT1 transporter. This effect
can occur with salicylic acid, and it is very pronounced with pyrazinoic acid and
its 5-hydroxy derivative, the metabolites of the tuberculostatic drug pyrazinamide.
Treatment with pyrazinamide, which is carried out over several months and at
dosages of gram amounts per day, may therefore trigger gout.
16.5 Explain the mode of action of uricosuric drugs.
is used right from the start and impinges on a far greater number of malignant
cells in the body. As a preventive measure, leukemia or lymphoma patients that
undergo chemotherapy receive allopurinol concomitantly. This treatment has
greatly reduced the incidence of acute urate nephropathy.
16.6 Explain the role of xanthine oxidase in purine degradation, and the therapeutic use
of xanthine oxidase inhibitors in gout and in tumor lysis syndrome.
16.7 Would uricosuric drugs be useful in the treatment of tumor lysis syndrome? Why, or
why not?
Adenine Hypoxanthine
Allopurinol
Guanine Xanthine
Urine
Uric acid
Rasburicase
Allantoin
Notes: This slide shows the action modes of allopurinol and of rasburicase, which
is an uricase enzyme from an Aspergillus mold that is recombinantly produced
in baker’s yeast (Saccharomyces cerevisiae). The enzyme will convert uric acid
to allantoin (see slide 16.5.5). This mode of action complements the decreased
formation of uric acid attained with allopurinol. Like the latter drug, rasburicase
is used—by way of intravenous infusion—to prevent acute urate nephropathy in
leukemia patients undergoing chemotherapy. Individually, rasburicase is superior
to allopurinol, but it should also be possible to use both drugs in combination.
Since rasburicase is a non-self protein, it is immunogenic, and it thus may
induce the formation of antibodies that inactivate it and additionally may cause
allergic complications. This problem is mitigated by the immunosuppressive effect
of chemotherapy and, in leukemia, of the disease itself. However, it will likely make
rasburicase unsuitable for long-term use in gout patients [112].
Notes: In contrast to the purine rings of AMP and GMP, which are assembled atop
the phosphoribose moiety, the skeleton of the pyrimidine bases is synthesized
before attachment to ribose phosphate.
306 16 Nucleotide metabolism
bicarbonate carbamoylphosphate carbamoylaspartate
O
OH
gln + 2ATP NH2 aspartate H2 N
O
1 2 O
HCO–3 O O P O− O N
H
O− OH
glu + 2ADP + Pi Pi
3
H2 O
orotidine mono-
phosphate
O PRPP O NAD+ O
5 4
HN HN HN
O O O
O N PPi O N NADH+H+ O N
H H
P OH OH OH
O
orotate dihydroorotate
OH OH
O O NH2
HN HN N
O gln + ATP
O N O N O N
P O OH P O P O
CO2 glu + ADP + Pi
OH OH OH OH OH OH
Notes: From OMP, it is only a short way to the final pyrimidine nucleotides. Decar-
boxylation of OMP yields UMP directly; glutamine donates another nitrogen in the
synthesis of CMP from UMP.
16.7 Synthesis and degradation of pyrimidines 307
CMP cytidine
dihydrouracil
Pi H2 O
O
NH3 NADPH+H+
HN
UMP uridine uracil
O N
Pi Pi ribose-1 P NADP+ H
H2 O
acetyl-CoA HO HO H2 N
Notes: Like their synthesis, degradation of pyrimidine bases is also fairly straight-
forward. Cytidine is deaminated to uridine. Pyrimidine nucleoside phosphorylase
cleaves uridine to free uracil and ribose-1-phosphate. One of the amide bonds in
the uracil ring is opened through hydrolysis, and ammonia and CO2 are cleaved
off to produce β-alanine. This compound may be excreted with the urine, or it
may be transaminated and converted to acetyl-CoA.2
Thymine is degraded in the same manner as uracil, producing methylated
analogues each step along the way. In the final step, methylmalonyl-semialdehyde
gives rise to propionyl-CoA, which is utilized as outlined in slide 10.3.6.
16.8 Explain the degradation of pyrimidine nucleosides.
10 no treatment
histidine
Surviving mice
O 8 irradiation,
H
N 6 carnosine
OH
4
O NH N
irradiation,
2
no carnosine
0
NH2 β-alanine
0 5 10 15 20 25 30
Days after irradiation
·OH
O O
H
N OH N
OH · OH ·
O NH N O NH N
HOH
NH2 NH2
DNA
TS
dUMP dTMP
uracil still has to be converted to thymine for the purpose of DNA synthesis. In
order to prevent incorporation of uracil into DNA, dUDP is first dephosphorylated
to dUMP by a cognate phosphatase. dUMP then enters the thymidylate synthase
reaction (TS); the product, dTMP, is phosphorylated twice to produce dTTP.
16.9 Explain the role and synthetic pathway of deoxyribonucleotides.
H H H R
H2 N N N H2 N N N H2 N N N HN
HN R
HN ⊕ HN HN
N N N H
O CH2 N R O H+ O H
H
O CH2 O
O
CH2
NH2 HN H NH2 HN NH+
3
HN
⊖S
Enzyme
O N S Enzyme O N S Enzyme
O N
dR dR
dR
Notes: Thymidylate synthase acquires a single carbon from the cosubstrate N,N ′
-methylenetetrahydrofolate (methylene-THF) and transfers it to dUMP. The reduc-
tion of the methylene carbon to the methyl group as found in the product (dTMP)
occurs at the expense of THF, which is thereby converted to dihydrofolate and has
to be reduced again to THF by dihydrofolate reductase (see slide 15.2.2).
Note that, halfway through the reaction, the enzyme, the substrate, and the co-
substrate are all joined together in a covalent intermediate, which is then resolved
through the abstraction of a proton from position 5 of the uracil by a catalytic
base in the enzyme’s active site (red arrow). This mechanism forms the basis of
the inhibition of thymidylate synthase by the antitumor drug 5-fluorouracil (see
slide 16.9.1).
same enzymes as noncancerous cells do; antimetabolites that kill cancer cells are
invariably toxic to noncancerous cells also. However, cancer cells are often more
susceptible to these cytotoxic drugs because they are more readily driven by them
into apoptosis, that is, programmed cell death (see slide 18.5.1), which is triggered
by a various forms of DNA damage.
As will become clear from the discussion of individual examples below, some drugs
can cause interference by more than one mechanism.
O N O N O N
5-F-dUDP
Thymidylate
Mutagenesis
synthase
dTMP DNA
Notes: 5-fluorouracil (5-FU) is a base analogue that mimics both uracil and thymine.
The dual resemblance endows it with a twofold mode of metabolic activation and
of action. One of several initial activation products, 5′-fluorodeoxyuridine, is also
used as a drug itself.
The metabolic activation of 5-FU occurs along the so-called salvage pathways
that recycle bases and nucleosides released by nucleic acid degradation. 5-FU is
activated by the same enzymes that salvage uracil. A key activation product is
5-FdUMP, which is a suicide substrate for thymidylate synthase (see next slide).
This is its major mode of action and the one that makes it an antimetabolite.
16.9 Nucleotide antimetabolites as anticancer and antiviral drugs 311
5-FU is also activated by enzymes that salvage thymine and thus may wind up
as 5′-fluoro-dUTP, which is incorporated into DNA; this will cause point mutations
(see slide 16.9.3).
HN NHR
N
N,N′ -methylene-THF
O
O O O CH2
F F
HN HN HN F NH2
O N O N O N S Enzyme
H
P O P O
thymidylate
synthase
OH OH
Adenine Guanine
N H N
dR N N dR N O
H
H
N N O N N O
H
H F F
N N N
H H
O N O N
dR dR
Notes: 5-F-dUTP can be incorporated into DNA and promote point mutations. This
is due to the fluorine, which withdraws electrons from the ring; this, in turn, pulls
electrons into the ring from other substituents and encourages the molecule to
312 16 Nucleotide metabolism
assume the iminol configuration. In this configuration, the base no longer pairs
with adenine but with guanine.
If the iminol configuration is present during DNA replication, guanine will be
chosen for incorporation into the opposite strand. This mutagenic effect of 5-FU
augments its anticancer effect.
NADPH+H+ NADP+
DHFR
H H
H2 N N N H2 N N N
HN HN
N N
H
O HN R O HN R
dTMP serine
TS SHMT
H
dUMP H2 N N N glycine
HN
N
O H2 C N R
suitable than most base analogues in antiproliferative therapy not related to tu-
mors. In particular, methotrexate is used to suppress lymphocyte proliferation in
severe autoimmune diseases such as rheumatoid arthritis or Crohn’s disease.
16.10 Explain the synthetic pathway for deoxythymidine triphosphate (dTTP), starting
with UDP. Explain how 5-fluorouracil and methotrexate interfere with this pathway.
mercaptopurine S Ribose-5-phosphate
N
NH
N N PRPP
H
PRPP
PRA
HGPRT
thio-IMP S IMP
N
NH
O−
−O P O N N
XMP Adenylosuccinate
O
O
OH OH GTP ATP
Notes: Cytosine arabinoside (cytarabine, araC) deviates from the normal cytosine
nucleotides not in the base but in its sugar moiety, arabinose, which is epimeric to
314 16 Nucleotide metabolism
ribose at the second carbon atom. Like ribonucleosides, cytosine arabinoside can
pass across the intestinal mucosa with the help of nucleoside transporters, which
facilitates its uptake.
N N N
HO N O HO N O HO N O
O O O
HO
OH OH OH OH
Notes: AraC enters the target cell by facilitated diffusion through the equilibrative
nucleoside transporter (ENT). In order to become a substrate for DNA polymerase,
araC must be phosphorylated to the triphosphate (araCTP); this is accomplished
by nucleoside and nucleotide kinases.
MDR ENT
dC deaminase
araU araC
5 ′-nucleotidase dC kinase
The activation of araC to araCTP can be intercepted at several stages. The extrusion
of araC itself is mediated by multi-drug resistance transporters (MDR) such as P-
glycoprotein, which belong to the family of ABC transporters (see slide 11.4.5).
Like cytidine and deoxycytidine, araC can undergo deamination either as a free
nucleoside or as a monophosphate, and the initial phosphorylation can be undone
by the enzyme 5′-nucleotidase. Increased expression of MDR or of enzymes that
counteract the activation of araC to araCTP cause tumor cell resistance (see slide
16.9.9).
16.9 Nucleotide antimetabolites as anticancer and antiviral drugs 315
100
no 5-NT overexpression
50
25
5-NT overexpression
0
0 12 24 36 48 60
Time (months)
which cleaves the DNA double strand [117]. Resealing of the double strand may
fail and lead to chromosome breaks.
−O P O O N O
O NH2
NH2
O N
N N N
−O P O O N N N
HO N
O O
O
O H3 C
NH
!
−O P O O N O
DNA O 2,3-dideoxy-adenosine (ddA)
OH
Notes: Base and nucleoside analogues are also useful in the treatment of viral
diseases. An example is dideoxyadenosine (didanosine, ddA), which lacks the 3-
OH group of the deoxyribose moiety. Since the 3- and the 5-OH groups form the
bonds within the DNA strand, incorporation of ddA into a growing DNA strand
causes termination of synthesis.3
The cellular DNA polymerases do not efficiently incorporate ddA, so it has
limited cytotoxicity. However, the reverse transcriptase enzymes in retroviruses
such as HIV use ddA efficiently, which leads to disruption of viral DNA synthesis.
Inhibitors of reverse transcriptase such as ddA are a standard component of HIV
therapy.
As stated above (slide 16.6.3), long-term application of didanosine may pro-
mote the manifestation of gout.
aciclovir ganciclovir
O O
N N
NH NH
HO N N NH2 HO N N NH2
O O
OH
Notes: Aciclovir (or acyclovir) and ganciclovir are even more severely crippled
than dideoxyadenosine, and it is quite remarkable that some enzymes even accept
3
You may recall that base-specific chain termination by dideoxy-nucleotides is the basis of the
Sanger method of DNA sequencing. If you don’t, consider yourself reprehended.
16.9 Nucleotide antimetabolites as anticancer and antiviral drugs 317
them as substrates. This is the case with enzymes of viruses from the Herpes
group, which includes herpes simplex virus, cytomegalovirus, varicella zoster virus
and a bunch of others. These are relatively large and complex viruses, which
contain not only the bare minimum of enzymes such as nucleic acid polymerases
but also their own nucleotide kinases.
Aciclovir Deoxyguanosine
O O
N NH N NH
HO O N N NH2 HO O N N NH2
O O
N NH N NH
P O O N N NH2 P O O N N NH2
cellular kinases OH
O O
N NH N NH
P P P O O N N NH2 P P P O O N N NH2
Notes: The selective toxicity of aciclovir for Herpes virus-infected cells, as opposed
to normal cells, arises at two stages. Firstly, aciclovir is phosphorylated to the
monophosphate only by viral nucleoside kinase, but not by the cellular enzyme.
Secondly, the triphosphate, which is formed from the monophosphate by cellular
kinases, is accepted as a substrate only by the viral DNA polymerase. This dual
mechanism of selective toxicity causes aciclovir to be well tolerated yet effective.
16.11 Explain the modes of action of the antiviral drugs dideoxyadenosine and aciclovir.
N
N N
N O
O N N O
O
P O O O
OH
O− O O O O
O
−O P
O O− O
O P OH
OH
Nucleic acid polymerases contain binding sites for the pyrophosphate (PPi ) that
is formed in the first step. Foscarnet lodges into the pyrophosphate binding
sites on several viral polymerases. Unlike pyrophosphate, foscarnet is resistant
to hydrolysis; it therefore remains bound to the polymerase enzymes and inhibits
their activity.
Cidofovir is similar to aciclovir in having a mutilated ribose moiety. In ad-
dition, it also carries a phosphonate group; it therefore functionally resembles a
nucleoside monophosphate and does not require the initial phosphorylation step,
which gives it a broader spectrum than aciclovir. After addition of two more phos-
phates to this phosphonate group by cellular kinases, the drug inhibits several
viral DNA polymerases.
Tenofovir disoproxil follows the same principle as cidofovir. However, it
additionally carries two ester groups on the phosphonate. These groups make the
drug more hydrophobic, which facilitates its diffusion across cell membranes. This
improves the efficiency of intestinal uptake and distribution within the body. After
cellular uptake, the ester groups are cleaved by esterases, which traps the molecule
inside the cell and allows it to interact with nucleotide kinases and polymerases.
Adenine and guanine nucleotides also inhibit the committed step (PRA synthesis), and IMP
inhibits the formation of PRPP from ribose phosphate.
16.3: Ribose can be converted to glucose in the HMS; therefore, 1 mole of ribose will
be converted to 5/6 moles of glucose. Deoxyribose, as its 5-phosphate, is cleaved by a
cognate aldolase to glyceraldehyde-3-phosphate and acetaldehyde. The former can enter
glycolysis or gluconeogenesis, whereas the latter is converted to acetyl-CoA, which in
human metabolism cannot be converted to glucose.
Two molecules of glyceraldehyde-3-phosphate are required to make one molecule of
glucose; therefore, the maximal yield of glucose would be 1 molecule of glucose for 2 mole-
cules of deoxyribose. Therefore, ribose provides 5/3 times more glucose than deoxyribose.
16.4: Dietary DNA and RNA are broken down by pancreatic DNAse and RNAse, respec-
tively. After dephosphorylation, the nucleosides are taken up by specific transport and
degraded in the liver. The sugars are cleaved by phosphorylases, which yields deoxyribose-
and ribose-1-phosphates. Purines are broken down to uric acid and excreted; pyrimidines
are broken down mostly to β-alanine and either excreted as such or completely degraded to
acetyl-CoA. The sugar 1-phosphates are first converted to 5-phosphates by pentose phos-
phate mutase and utilized by the hexose monophosphate shunt (ribose-5-phosphate) or
by cleavage to glyceraldehyde-3-phosphate and acetaldehyde (deoxyribose-5-phosphate).
16.5: Uricosuric drugs competitively inhibit the tubular reuptake of uric acid by the URAT1
transporter. This accelerates the renal excretion of uric acid.
16.6: In purine degradation, adenine and guanine are converted by deamination to hypox-
anthine and xanthine, respectively. Xanthine oxidase converts hypoxanthine to xanthine,
as well as xanthine to uric acid. Uric acid is the final product of purine degradation and is
excreted.
Uric acid has limited solubility, and increased levels of uric acid lead to its precipi-
tation or crystallization in joints (gout) or in the kidneys. The latter occurs most acutely
and dramatically in tumor lysis syndrome during chemotherapy; this is most common in
leukemia. In both conditions, inhibition of xanthine oxidase prevents the final oxidation
steps, leading to the excretion of hypoxanthine and xanthine of uric acid. These are more
soluble than uric acid and therefore less prone to precipitate prior to excretion.
16.7: In tumor lysis syndrome, uricosuric drugs would only further accelerate the buildup
of uric acid inside the kidney tubules and would make matters worse.
16.8: Cytidine is deaminated to uridine by cytidine deaminase. Uridine is cleaved by a
phosphorylase to uracil and ribose-1-phosphate. The uracil ring is then opened through
hydrolysis, and cleavage of ammonia and CO2 gives β-alanine. The latter can be further
degraded by transamination and dehydrogenation to acetyl-CoA, or alternatively it can be
used for the synthesis of carnosine.
The degradation of thymidine is analogous and produces methylated analogs, with
propionyl-CoA as the final product.
16.9: Deoxyribonucleotides function exclusively as precursors of DNA. Deoxyribonucle-
oside diphosphates—dADP, dCDP, dGDP and dUDP—are formed from the corresponding
ribonucleoside diphosphates by the same enzyme, ribonucleotide reductase. All except
dUDP are then phosphorylated to the triphosphates.
In contrast, dUDP first undergoes dephosphorylation to dUMP and is then converted
by thymidylate synthase to dTMP. The latter is then phosphorylated to dTDP and finally
to dTTP.
16.10: UDP is converted to dUDP by ribonucleotide reductase and then dephosphorylated
to dUMP. Thymidylate synthase transfers the C1 from N,N ′-methylenetetrahydrofolate to
320 16 Nucleotide metabolism
position 5 of the uracil base in dUMP, producing dTMP. This reaction proceeds through
an intermediate state in which methylene-THF, uracil and the enzyme are all covalently
linked. Subsequently, dTMP is phosphorylated twice to dTDP and then dTTP.
5-Fluorouracil is activated to 5-dUMP via salvage reactions. It functions as a substrate
analogue for thymidylate synthase. The reaction stalls at the stage of the covalent reaction
intermediate; since the enzyme is unable to abstract fluorine from the uracil ring, it
remains trapped in the covalent intermediate.
In the thymidylate synthase reaction, N,N ′-methylenetetrahydrofolate is converted
to dihydrofolate, which needs to be reduced by dihydrofolate reductase before it can
acquire another C1 subunit and supply it to thymidylate synthase. Methotrexate inhibits
dihydrofolate reductase and therefore indirectly inhibits dTMP synthesis.
16.11: Dideoxyadenosine lacks the 3-OH group in the ribose moiety to which the next
nucleotide would be attached in DNA synthesis. Therefore, its incorporation into a grow-
ing DNA strand will cause synthesis to terminate. It is not an efficient substrate for
human DNA polymerase but is readily accepted by retroviral reverse transcriptase, and is
accordingly used in the treatment of HIV infections.
Aciclovir is used to treat herpes virus infections. It has a rudimentary sugar moiety in
which the carbons 2 and 3 of ribose are altogether lacking. It is not an efficient substrate
of human nucleoside kinases, yet it is phosphorylated by herpes virus nucleoside kinase.
Moreover, the triphosphate inhibits herpes virus DNA polymerase but not the human
enzyme.
Chapter 17
N⊖ N
Fe2⊕
⊖
N N
HO
Notes: Heme consists of a porphyrin ring that holds a central iron ion. “Porphyr”
is the name of purple-colored gemstone. Heme is colored, too, but its color varies
with the protein it is bound to and with the oxidation state of the iron. In addition
to hemoglobin, heme is also found in many cytochromes, a term that literally
translates as “cell color” and comprises various classes of enzymes and electron
carriers. The red-brown colors of tissues like liver, kidney, and brown fat are due
to their abundance of heme-containing enzymes or of mitochondria, which contain
many heme cofactors within the proteins of the electron transport chain.
In this chapter, we will focus on the biosynthesis and degradation of heme
itself. The roles of heme in the respiratory chain and in cytochrome P450 enzymes
are covered in chapters 6 and 18, respectively.
321
322 17 Iron and heme metabolism
Redox chemistry
• electron transport: cytochromes in the respiratory chain
• enzyme catalysis: cytochrome P450, cyclooxygenase, others
Reversible binding of gases
• O2 : hemoglobin and myoglobin (80–90% of all heme)
• NO: guanylate cyclase
Notes: In hemoglobin and myoglobin, the heme iron remains in the ferrous or
Fe2+ state throughout the cycle of oxygen binding and release. In redox-active
enzymes and in the respiratory chain, heme regularly goes back and forth between
the ferrous and the ferric or Fe3+ states, and sometimes also the ferryl or Fe4+
state.
In cytochrome P450 enzymes, the function of heme is to facilitate the forma-
tion of reactive oxygen (see slide 18.2). Similarly, formation of reactive oxygen
species (ROS) may also occur as a side reaction wherever heme functions in trans-
port of oxygen or of electrons, and protective mechanisms are required to contain
damage by ROS. In hemoglobin, molecular oxygen (O2 ) may abscond with an extra
electron and thereby turn itself into superoxide (O–2 ), while the heme iron is left
one electron short. Hemoglobin that has lost one electron is called methemoglobin;
it is unable to carry oxygen. Its iron is reduced to the ferrous form again by an
NADH-dependent enzyme, methemoglobin reductase.1
Protective mechanisms that scavenge reactive oxygen species include the en-
zymes superoxide dismutase and catalase, as well as antioxidants such as to-
copherol (vitamin E; see slide 11.6.11) and ascorbic acid (vitamin C). Organisms
that lack such protective mechanisms are anaerobic, that is, they survive only in
the absence of oxygen. Anaerobic organisms occur among both prokaryotes and
eukaryotes.
17.1 Summarize the biochemical and physiological functions of heme.
17.2.1 Overview
Notes: Heme contained in the food is taken up from the intestines, but this is
only relevant because of the iron it contains. The organic porphyrin ring is mostly
synthesized from scratch. The synthetic pathway is split across two compartments;
1
Methemoglobin reductase also participates in reductive metabolism of drugs and xenobiotics
(see section 18.4). In that context, it is often referred to as diaphorase.
Interestingly, methemoglobin binds cyanide ions more avidly than reduced hemoglobin does. This
is used in the treatment cyanide poisoning: Controlled application of oxidizing agents—such as
dimethylaminophenol or sodium nitrite, [118]—turns some hemoglobin into methemoglobin, which
then captures cyanide and prevents its binding to hemes in the respiratory chain. It seems, however,
that treatment with hydroxycobalamin is superior (see slide 15.5.5)
17.2 Heme biosynthesis 323
the initial and final steps occur in the mitochondria, while the intervening ones
occur in the cytosol. Heme exerts feedback inhibition on the first step in the
pathway.
mitochondria cytosol
8 succinyl-CoA + 8 glycine
synthesis of
building blocks
8 δ-aminolevulinic acid 4 porphobilinogen
formation of
linear precursor
hydroxymethylbilane
ring closure
protoporphyrinogen uroporphyrinogen
ring modifications
protoporphyrin coproporphyrinogen
iron emplacement
heme
Notes: The first reaction in the porphyrin synthesis pathway occurs in the mi-
tochondria. Glycine forms a Schiff base with pyridoxal phosphate (PLP) in the
aminolevulinate synthase enzyme. Pyridoxal phosphate acts as a transient “elec-
tron sink”, as it usually does and as is illustrated for serine hydroxymethyltrans-
ferase in slide 15.2.4. Decarboxylation of the enzyme-bound glycine produces a
carbanion intermediate, which reacts with the carbonyl carbon in succinyl-CoA.
OH OH OH
O O O
O⊖ PLP H2 O H2 O
⊖
O CH2 O O O
N S
H2 N CO2 PLP CoA CoA-S– N PLP NH2
PLP
Lys NH2
porphobilinogen H+
Enzyme
HO HO
Lys NH2
HO O HO O
O O
Lys N N Lys
H H
N N
H H
NH2 NH2
HO O
O
NH2
HO
NH2
Notes: This pretty picture (rendered from 1pv8.pdb) shows a model compound
(structure on the right) inside the active site of porphobilinogen synthase, next
to a zinc ion. It sheds little light on the reaction mechanism, or on the specific
role of zinc. The only point I want to make here is that zinc is important for this
enzyme. In lead intoxication, the zinc in porphobilinogen synthase is dislodged
17.2 Heme biosynthesis 325
by lead, which inhibits the enzyme and therefore the biosynthesis of heme and
hemoglobin.
17.3 What is the effect of lead intoxication on heme biosynthesis, and what laboratory
findings would you expect in patients suffering from it?
H2 N
Ac P Ac P N Ac P Enzyme-SH Ac P
H
Enzyme S Enzyme S H2 N
N N N N
H H H H
NH3 NH3
Ac P
4 H2 N
N 4 NH3 Ac P Ac P Ac P Ac P Ac P Ac P
H
Enzyme S
N N N N N N
H H H H H H
H2 O
Ac P Ac P Ac P Ac P
HO
N N N N
hydroxymethylbilane H H H H
hydroxymethylbilane Ac P
Ac P Ac P Ac P Ac P N
N H Ac
HN
HO
N N N N H P
H H H H Pr N
Ac
P Ac
coproporphyrinogen III
P Ac P
N N
H Ac H Ac
NH HN NH HN
P H P P H P
N N
4 CO2
P P Ac uroporphyrinogen III
P
O2
N N
H H
NH HN NH HN
P H P 2 CO2 + 2 H2 O P H
N N
3 O2
P P
3 H2 O2
protoporphyrin IX heme
⊖
N N
H Fe++
⊕⊕
N N N Fe N
P H P
N N
⊖
2 H+
P P
Notes: Regardless of the underlying cause, the inhibition of heme synthesis will
result in red blood cells that are smaller and contain less hemoglobin than normal
ones. This condition is named microcytic, hypochromic anemia. The term “micro-
cytic” refers to the reduced cell size, whereas “hypochromic” denotes the reduced
intracellular hemoglobin concentration.
17.5 Explain the connection between vitamin B6 deficiency and microcytic anemia.
328 17 Iron and heme metabolism
Ac P P
N N
Ac H Ac H
NH HN NH HN
P H P P H P
N 4 CO2 N
P Ac Ac P P
N
Ac H Ac
N N
P H P
N
spontaneous
oxidation uroporphyrin III
P Ac
Absorbance
0
350 400 450
Wavelength (nm)
The absorbed wavelength range (or absorption spectrum) differs between the var-
ious porphyrins. Uroporphyrin III has an absorption peak at 405 nm, which is at
the blue end of the visible spectrum; this peak is readily detectable in blood serum
samples (left). The sun light is more intense in the visible range than in the UV
range. Sun screen lotion, which is designed to absorb UV light but not visible light,
will not prevent photosensitization by uroporphyrin III.
Notes: Red blood cells have a regular lifespan of 120 days (although it can be
considerably shorter in some diseases). At the end of this lifespan, they are cap-
tured and ingested by phagocytes in the spleen and the liver. When the globin
protein is proteolytically degraded, heme is released. Heme itself undergoes degra-
dation mostly in the liver. Ring cleavage by heme oxygenase produces biliverdin,
which is in turn reduced to bilirubin. Some bilirubin is excreted into the bile
as such; however, the greater share is first conjugated with glucuronic acid by
UDP-glucuronosyltransferase, form 1A1, and excreted thereafter. The major trans-
port protein responsible for excretion of the diglucuronide is an ABC transporter
(ABCC2), the same one that also secretes bile acids (see slide 11.5.3).
In the large intestine, part of the conjugated bilirubin undergoes deconjuga-
tion by bacterial β-glucuronidases. In the anaerobic environment that prevails
2
Generally speaking, any kind of psychiatric disturbance can potentially be due to organic causes,
and therefore a thorough medical history and examination is necessary in each psychiatric patient. If
you decide to become a psychiatrist, make it a point not to forget about your neurology and internal
medicine.
17.4 Heme degradation 331
inside the colon, the released bilirubin subsequently undergoes reduction, again
by bacterial enzymes, to variously colored pigments that produce the stool color.
Another reduction product, urobilinogen, is taken up and excreted with the urine,
causing the yellow color of the latter.
HO N N
H H
3NADPH + 3O2 NH HN
O O
CO + Fe3+ + 3NADP+ + 3H2 O
HO O O OH 2 UDP
O
N O
H NADPH 2 UDP-glucuronide
N HN
O H
N N N
NADP+
H H
HO O NH HN
HO O O bilirubin
biliverdin
17.8 Explain how heme is degraded, and how the degradation product is disposed of.
17.4.1 Jaundice
Liver diseases can affect synthesis, conjugation and biliary secretion of biliru-
bin in various degrees, and either form of bilirubin can be more strongly increased
than the other.
Notes: This slide shows a brain section from a patient with severe bilirubin en-
cephalopathy. The yellow color in the deeper structures of the forebrain—the
so-called basal ganglia—is due to bilirubin accumulation. Through an unknown
biochemical mechanism, bilirubin causes damage to the basal ganglia, which re-
sults in motor dysfunction and other neurological symptoms.
3
Prior to birth, the bilirubin formed by the fetus is eliminated via the placenta and therefore does
not accumulate. The same applies to the toxic metabolites that accumulate in phenylketonuria (see
slide 12.5.1) and several other hereditary metabolic diseases, which therefore become manifest only
after birth.
17.4 Heme degradation 333
HO O O OH HO O O OH
4Z,15Z 4E,15Z
N N O N N N
H H H H H
NH HN HN
O O O
HO O O OH HO O O OH
4Z,15E
N N N O N N
H H H N H H
NH O HN
O O
15Z-lumirubin
While the absorption maximum of bilirubin is in the blue wavelength band, green
light reportedly produces lumirubin more efficiently, and it also induces less cyto-
toxic byproducts in cell culture models [123]. It seems, however, that blue lamps
are still more widely used in practice, and the literature does not make mention
of significant side effects of blue light, even in the long-term treatment of Crigler-
Najjar patients [124].
17.9 Explain the rationale and application of phototherapy in newborns.
334 17 Iron and heme metabolism
Sn-mesoporphyrin
HO 30
Phototherapy
25
O
20
Percent
N⊖ N
15
Sn++
⊖ 10
N N
5
O
0
0 5 10 15 20 25
HO
Peak plasma bilirubin (mg/dL)
Notes: The inhibition of heme oxygenase with Sn-mesoporphyrin has been used
successfully in clinical studies to treat neonatal jaundice. The study summarized
in this slide examined the effectiveness of Sn-mesoporphyrin in the treatment of
newborns with glucose-6-phosphate dehydrogenase deficiency (see slide 9.4). In
this condition, the lifespan of red blood cells is diminished, which increases the
rate of heme degradation; newborns therefore are at an increased risk of severe
jaundice.
Remarkably, a single injection of the drug was sufficient to reduce the peak
levels of bilirubin to a greater extent than the reference treatment (phototherapy).
It should be noted that both forms of treatment sufficed to keep the peak bilirubin
levels below 25 mg/dL, which is considered sufficient to avoid damage to the brain.
Phototherapy currently remains the standard treatment in clinical practice. Figure
prepared from original data in [125].
heme molecule. Binding of NO to one face of the heme releases a histidine side
chain on the other, which causes a conformational change and activation of the
sGC molecule.
In vitro experiments show that CO can also bind and activate sGC. It has there-
fore been proposed that heme oxygenase, which produces CO, has a regulatory
role in addition to its metabolic one. However, while this idea has been around for
awhile, I have not come across solid evidence that supports a significant signaling
role of CO in vivo.
Notes: In diseases that cause iron overload, the storage capacity of ferritin will be
exceeded. Excess iron will then precipitate around the ferritin particles and cause
them to aggregate. These iron-rich aggregates are called hemosiderin.
deaminase. Its episodes can be triggered by drugs that induce ALA synthase. Excretion of
porphobilinogen causes the urine to turn red. Accumulation of ALA itself causes neuro-
logical and psychiatric symptoms due to competitive inhibition of GABA receptors in the
central nervous system.
17.8: Heme oxygenase breaks he porphyrin ring and releases CO. The product, biliverdin,
is then reduced to bilirubin. The two carboxylic acid moieties of bilirubin are conjugated
with glucuronic acid by UDP-glucuronosyltransferase, and bilirubin-diglucuronide is se-
creted with the bile.
17.9: In newborns, the enzyme activity of UDP-glucuronosyltransferase 1A1 is low, so that
unconjugated bilirubin accumulates. This poses a risk of neurological damage.
The unconjugated bilirubin distributes throughout the body, including the skin, and
in the latter location is accessible to light. Absorption of blue or green light promotes the
formation of bilirubin isomers that are more readily excreted even without conjugation.
Phototherapy therefore lowers the level of bilirubin.
17.10: Ferritin is the major intracellular storage medium for iron. Each ferritin particle is
a large protein complex with 24 identical subunits. It forms a hollow shell that can store
up to 4,500 iron ions.
Chapter 18
339
340 18 Metabolism of drugs and xenobiotics
O NADPH+H+ NADP+ O OH
H H
N N
O O
N N
H O2 H2 O H
O O
UDP-glucuronide PAPS
UDP 3′ -P-AMP
O
OH
O O S O−
O OH O
H O H
N N O
OH
O −O O
O
N N
H H
O O
Notes: Drug metabolism works hand in hand with excretion. The key organs in
drug metabolism and excretion are the small intestine, the liver, and the kidneys.
Drug-modifying enzymes are highly expressed in the small intestine and the
liver. The reactions catalyzed by these enzymes are classified into phase I and
phase II reactions. Broadly speaking, a phase I reaction introduces a functional
group into a substrate that enables its subsequent conjugation in a phase II reac-
tion. Conjugation, in most cases, increases the polarity of the substrate, rendering
it more amenable to secretion into the bile or to excretion in the urine. The two
stages of phenobarbital metabolism depicted in the preceding slide exemplify
phase I and phase II transformations, respectively.
18.2 Cytochrome P450 enzymes 341
Drug molecule
phase 1
phase 2
Reactive metabolite Conjugate
Urine
Deconjugation,
reuptake
ER membrane
Notes: Cytochrome P450 is a large family of enzymes that are found in both
prokaryotic and eukaryotic organisms. In mammalian cells, they are found in both
the ER and the mitochondria, and they function in various biosynthetic pathways,
such as cholesterol and steroid hormone synthesis. Several dozen CYP450 (CYP)
isoforms are involved in drug metabolism; they are the most important group of
enzymes in phase I metabolism. These isoforms are found mostly in the ER.
The reactions catalyzed by CYP enzymes start with the abstraction of hydrogen
from NADPH by cytochrome P450 reductase, an auxiliary enzyme. The hydrogen
is used by CYP to reduce one of the two atoms of molecular oxygen to water. The
342 18 Metabolism of drugs and xenobiotics
other oxygen atom is retained in a highly reactive form, which is then used to force
one or the other kind of reaction on a substrate.
[O]
R2 N−H R2 N−OH Heteroatom oxidation
[O]
R3 N R3 N−
−O
[O]
R2 S R2 S−
−O
[O]
RO−CH2 R ROH + O−
−CHR Dealkylation
[O]
R2 N−CH2 R R2 NH + O−
−CHR
[O] O
R−HC−
−CH−R R−HC CH−R Epoxide formation
Notes: Apart from aromatic or aliphatic hydroxylation, CYP enzymes can also
bring about several other types of reactions. Oxidation can occur once or repeat-
edly; for example, hydroxyl groups can be further oxidized to aldehydes and car-
boxylic acids. Oxidation is not confined to carbons but can also affect heteroatoms.
Dealkylation is a powerful way to break up and inactivate drug molecules.
Amine oxidation, aldehyde formation, and epoxide formation yield reactive
groups that may subsequently cause toxic effects. Therefore, while drug metabo-
lism often abolishes toxicity, sometimes is can actually create it.
18.2 Explain the mode of action of cytochrome P450 enzymes, and name some typical
reactions that they perform on drug molecules.
Notes: Considering that there are several dozen different CYP enzymes in human
cells, it is remarkable that a single isoform, CYP3A4, is involved in the metabolism
of some 50% of all clinically prescribed drugs. Like several other isoforms, CYP3A4
is inducible, that is, its rate of gene transcription is increased by certain drugs.
With CYP3A4, the nuclear hormone receptor that mediates induction is the
pregnane X receptor (PXR). When a suitable drug binds to this receptor, it translo-
cates from the cytosol to the nucleus and binds to its cognate regulatory DNA
sequences, referred to as xenobiotic response elements (XRE); this results in in-
creased transcription of genes in the vicinity. The PXR responds to a particularly
wide variety of drugs, which in part accounts for the prominent role of CYP3A4 in
drug metabolism.
18.2 Cytochrome P450 enzymes 343
Cytochrome P450,
PXR phase II enzymes,
ABC transporters
HO
O
O O
HO O OH
O
O O
HO
O
OH
Notes: Another reason of CYP3A4’s prominent role in drug metabolism is its own
ability to accommodate a wide variety of substrates within its active site. This
slide and the next one illustrate two examples. The drugs, erythromycin in this
slide and ketoconazole in the next one, are shown in blue or green. The heme of
CYP3A4 is shown in red, and several amino acid side chains that interact with the
drug molecules are shown in yellow. Note the differences in protein conformation
and the interacting residues between both slides.
344 18 Metabolism of drugs and xenobiotics
Cl
Cl
O
N
N O
N O
N
Notes: This slide shows two molecules of ketoconazole bound within the active
site of CYP3A4. The binding of the second molecule is likely an artifact of the high
drug concentration used in the crystallography experiment.
Note that the imidazole group of one drug molecule is bound to the heme iron.
Ketoconazole is an antifungal agent that inhibits 14α-demethylase, a cytochrome
P450 enzyme that is essential for the synthesis of ergosterol, the major sterol
found in fungal cell membranes. One side effect of ketoconazole is the inhibition
of drug metabolism in humans.
Structures rendered from 2j0d.pdb and 2v0m.pdb, [126].
CYP450 Fexofenadine
HO
N N
H2 N O H2 N O
N HO
HO
N
Cl Cl
HO
CYP450
N N
N NH
HO
O O
CYP450
HO O
Diazepam Oxazepam
Notes: While drug metabolism often results in inactivation, metabolites may retain
pharmacological activity, or sometimes even acquire novel ones. For example,
two CYP-mediated reactions convert diazepam to oxazepam, which retains the
pharmacological activity of the parent compound.
Some active metabolites, including oxazepam and fexofenadine, have become
drugs in their own right. Since these molecules are already prepared for conju-
gation, they are usually more rapidly eliminated than the parent compounds. In
the case of oxazepam, this is an advantage when the intention is to induce sleep,
since most of the drug will have been excreted the next day. In contrast, diazepam
works better in the treatment of epilepsy, where a more stable and steady level of
drug activity is desired.
With all examples shown in this slide, both the parent molecules and the me-
tabolites have pharmacological activity. For various applications, drugs have been
designed that actually require metabolic conversion to become active; such mole-
cules are referred to as prodrugs. Some organic nitrates, for example nitroglycerin,
are metabolized by cytochrome P450 enzymes to release nitric oxide as the active
principle. Other examples of prodrugs are sulfonamides (see slide 15.3) as well as
tenofovir and cidofovir (slide 16.9.14).
18.5 Explain the concept of active drug metabolites.
1
In the molecular structures, there also are some discontinuities. These represent conforma-
tionally flexible segments of the polypeptide chain that did not give rise to distinct diffraction
signals.
346 18 Metabolism of drugs and xenobiotics
Benzopyrene
CYP1A1
DNA
O
OH
OH Benzopyrene–DNA adduct
Notes: Fortunately for smokers, not all aromatic epoxide molecules will end up
reacting with DNA. One major detoxification pathway is the reaction with glu-
tathione, which is facilitated by glutathione-S-transferase (left). Epoxide hydrolase
(right) also contributes to the detoxification.
OH
OH G−SH H2 O
OH
NH2
O
OH O NH
OH
S O
OH
NH
OH
OH
OH
O OH OH
The epoxide hydrolase reaction occurs after a phase I reaction, so therefore could
be considered part of phase II. On the other hand, it does not result in conjugation,
and it thus might be considered not to be part of phase II. So, which phase does it
belong to?
This question is purely one of definition and therefore irrelevant. It is only
mentioned to illustrate that the distinction between phase I and II reactions, while
useful, has its limitations.
348 18 Metabolism of drugs and xenobiotics
HN O
Acetaminophen NAPQI
G–SH
SG
HN O N O OH
CYP450
OH O HN O
Protein–SH
S Protein
OH
HO O OH −O
O O O OH
UDP
O
OH
Morphine Morphine-3-glucuronide
HO
OH
Notes: Phase I reactions are not necessary if a drug molecule already contains
functional groups suitable for conjugation. An example is morphine, which has
two hydroxyl groups. The conjugation of either, or both, with glucuronic acid is
sufficient for excretion. Interestingly, one of the two single glucuronides—the one
not shown here—retains pharmacological activity.
18.3 Phase II reactions 349
CoA-SH
N N O OH
25
Frequency (individuals)
20
15
10
0
0 1 2 3 4 5 6 7 8 9
INH in plasma (µg/mL) after 6h
Notes: The depicted experiment measured the speed of acetylation. A fixed test
dosage was applied at t=0, and the remaining plasma concentration was deter-
mined 6 hours later. There clearly are two separate peaks, which represent the
fast and slow acetylators, respectively. Among Caucasians, about 50% express an
inactive NAT2 allele, which causes the slow-acetylator phenotype. The percentage
of slow acetylators is lower among Asians (but was higher in a small study on
Kenyans [127]; I don’t know how representative that study is).
Apart from isoniazid, the NAT2 enzyme and its polymorphism also affects
the inactivation rates of some other drugs, such as procainamide and hydralazine,
which in slow acetylators are more likely to cause toxicity. A role of this polymor-
phism has also been reported in the susceptibility to bladder cancer caused by
aromatic amines, which in Europeans was found to correlate with slow acetylator
350 18 Metabolism of drugs and xenobiotics
status. Surprisingly, this correlation was not observed in Chinese [128]. The reason
for this discrepancy is unknown.
18.9 What are fast and slow acetylators?
Glutamine
H2 N O
Phenylacetic Conjugate
acid
H2 N O
O
H2 N
O O OH O
CoA–SH CoA O
OH S N
H
OH
Notes: Amino acid conjugation is limited to xenobiotics that are carboxylic acids,
which are first activated to coenzyme A-thioesters and then linked to an amino
acid by an amide bond. The amino acid may be glutamine (as shown), glycine, or
taurine. The latter two amino acids are also used in the synthesis of the conjugated
bile acids taurocholate and glycocholate (see slide 11.5.1).
The pathway can be put to use in the alternate pathway therapy of urea cycle
enzyme defects (see slide 12.3.10). In this application, the usual physiological
role of amino acid conjugation is turned on its head. Normally, amino acids are
expended in order to eliminate unwanted organic acids; in contrast, here we supply
innocuous organic acids, the amino acid conjugates of which then become vehicles
for the elimination of surplus nitrogen. Brilliant!
An organic acid commonly used in alternate pathway therapy is phenylbu-
tanoic acid, which first undergoes β-oxidation to phenylacetic acid (the substrate
shown here) before conjugation to glutamine and excretion. It may be combined
with benzoate, which undergoes conjugation with glycine and therefore recruits
another enzyme and another pool of nitrogen.
18.10 Name the major types of reactions, enzymes and cosubstrates in phase II drug
metabolism.
Multiple enzymes:
• methemoglobin reductase (diaphorase)
• cytochrome P450 reductase
• thioredoxin
• bacterial metabolism
• ...
18.5 Anti-tumor drugs that are preferentially activated in tumor cells 351
Notes: While cytochrome P450 enzymes play the most important role in phase I
metabolism, some drugs are actually reduced rather than oxidized at this stage.
The enzymes involved are a somewhat heterogeneous bunch and primarily serve
in roles other than the metabolism of xenobiotics.
This section, after some introduction, examines two antitumor prodrugs that at-
tempt to exploit metabolic activation to selectively target tumor cells.
Microtubule
Bcl family Cytochrome C Mitochondria
disruption
N N NH2
Cl–
dR
Cl
OH N OH N OH
⊕ ⊕ ⊕
N N N
N N N
H2 N N N N N NH2 N N NH2
dR dR dR
Notes: The experimental drug CB 1954 is, in its original form, a monovalent alky-
lating agent; the alkylating group is the aziridine group (the three-membered ring
at the top). It is activated to a bifunctional agent in two steps. Diaphorase (methe-
moglobin reductase) reduces the nitro group to a hydroxylamine, which is then
acetylated by an acetyltransferase to form a reactive acetoxy group [129].
acetyl-CoA
CoA–SH
N NH2
N NH
N DNA
⊕
H
N O
O
O N N N NH2
O acetate H
N
H2 N NO2 NH
N
O
O
H2 N NO2
For reasons that are not well understood, the environment inside tumor cells is
often more reducing than in nontumorous cells, which will result in preferential
activation of this drug inside tumor cells. CB 1954 is somewhat of a perennial
“experimental” drug, which probably means that it is not very compelling in clin-
ical use. However, several established anticancer drugs such as doxorubicin and
bleomycin also require reductive activation [130], which may enhance their selec-
tivity for cancer cells.
18.6 Answers to practice questions 353
OH
NH2
O
O⊖ Cl OH Cl Alkylating drug
H Cl Cl
O NH O N N
O ⊖O
O S P Cl P Cl
HO H N N
O O O
NH
O
Cl Cl
O
R S
Canfosfamide O Byproduct
Notes: Glutathione conjugation can inactivate alkylating agents, for example ones
with epoxide groups (slide 18.3.2) or anticancer drugs such as mechlorethamine
(slide 18.5.2). Accordingly, tumor cells may develop resistance to alkylating antitu-
mor drugs by increasing their expression of glutathione-S-transferase.
The antitumor agent canfosfamide targets such cells, since it has been de-
signed to require activation by glutathione-S-transferase (GST; the catalytic residue
is a tyrosine). This drug is currently in clinical trials.
The pregnane X receptor, or PXR, has particularly broad ligand specificity. PXR medi-
ates transcriptional activation of CYP3A4, which in turn participates in the metabolism of
∼50% of all drugs.
18.4: 1. CYP3A4 and several other enzymes have flexible active sites that can accommo-
date a large variety of substrates. 2. Cytochrome P450 enzymes transfer oxygen in a highly
activated form that can react with a wide range of different functional groups. Therefore,
it is not necessary for the substrate to interact or cooperate with the enzyme in a highly
specific manner.
18.5: Metabolic transformation of drug molecules does not always lead to inactivation,
and where it does not, the products are referred to as active metabolites. Some drugs even
require metabolic transformation for activity; these are referred to as prodrugs.
18.6: Polycyclic aromatic hydrocarbons are hydrophobic and planar molecules and, as
such, tend to intercalate into DNA. Since the unmodified aromatic compounds are chem-
ically inert, intercalation as such does little damage. However, introduction of epoxide
bonds by cytochrome P450 (in particular type 1A1) makes the aromatic hydrocarbons
reactive and enables them to covalently react with DNA. During DNA replication, such
covalent adducts cause mutations and potentially cancer.
Epoxides can be detoxified by phase II enzymes, in particular glutathione-S-transferase
and epoxide hydrolase.
18.7: After intercalation, a nucleophile in the DNA, typically a nitrogen, will perform
nucleophilic attack on one of the carbons in the epoxide. The reaction is analogous to the
one shown in slide 18.3.2, in which the sulfur of glutathione acts as the nucleophile.
18.8: Acetaminophen is converted by cytochrome P450 to a reactive intermediate (N-
acetyl-p-benzoquinone imine, NAPQI) that reacts with glutathione. Toxicity ensues when
glutathione is depleted, leaving the cell unprotected from excess NAPQI and reactive
oxygen species.
18.9: Fast and slow acetylators are individuals that carry different alleles for the enzyme
N -acetyltransferase 2 (NAT2). In slow acetylators, the enzyme is inactive. This causes a
slower rate of acetylation and inactivation of INH and several other drugs, particularly
aromatic amines.
18.10:
1. Glucuronic acid conjugation (UDP-glucuronosyltransferases, UDP-glucuronide)
2. Sulfate conjugation (Sulfotransferases, Phosphoadenosine-phosphosulfate)
3. Acetylation (N - and O-acetyltransferases, acetyl-CoA)
4. Methylation (Methyltransferases, S-adenosylmethionine)
5. Amino acid conjugation (glutamine, glycine, taurine)
Chapter 19
• How many organs are affected by the enzyme defect: One organ, a few, or all
organs?
• How severe is the defect?
• Can the defect be adequately controlled by conventional treatment?
Notes: While some enzymes, such as pyruvate dehydrogenase, are expressed and
required in almost all cells, others are expressed exclusively or preferentially in
selected organs. For example, UDP-glucuronosyltransferases are expressed mostly
in the liver, and enzyme defects accordingly affect this organ the most.
The clinical severity of enzyme defects ranges from benign to fatal. A fairly
benign defect is the Gilbert syndrome, in which the rate of bilirubin glucuronida-
tion is reduced. This condition requires nothing more than caution in the selection
and dosage of drugs when treating other diseases. On the other hand, untreated
adenosine deaminase deficiency is fatal (see sections 16.5 above and 19.2 below).
Obviously, aggressive therapeutic strategies such as organ transplants or gene
therapy are warranted only with severe defects.
• diets
• drugs
• organ transplants
Notes: Diets can be used in many enzyme defects, and if effective, they are cer-
tainly preferable. An example is the fructose- and sucrose-free diet in fructose
intolerance, which effectively prevents the deleterious depletion of free phosphate
and of ATP in this disease (see slide 4.2.2).
355
356 19 Enzyme and gene therapy of enzyme defects
Drugs are useful in several hereditary enzyme defects, but usually in a limited
way. We have already discussed the use of NTCB in tyrosinemia (slide 12.5.4) and
of organic acids in urea cycle defects (slide 12.3.10); a few more examples are
discussed below. Organ transplants are useful in those diseases that primarily
affect a single organ, typically the bone marrow or the liver.
19.1 Explain how organ transplants can be used to treat genetic enzyme defects.
Correction of . . .
• DNA: gene therapy
• mRNA: suppression of mutant stop codons with drugs
• protein: enzyme substitution
Notes: Enzyme defects are in principle the most logical and suitable targets for
gene therapy. A key advantage of gene therapy is its long-lasting effect—at least
in principle, the treatment maybe be effective for life. However, in most enzyme
defects, gene therapy is still at the experimental stage, and it has not yet become
the standard therapy in any specific disease.
Enzyme substitution therapy is more widely used. Since the enzyme molecules
have a limited half-life, the enzyme has to be applied regularly, often in weekly
or bi-weekly intervals. While this is costly and somewhat inconvenient, it has
the advantage that the therapy can simply be discontinued in case of untoward
reactions, which would obviously not be feasible with gene therapy.
Correction at the mRNA level through suppression of stop codons—also re-
ferred to as translational antitermination—is confined to a few specific examples;
obviously, not all gene defects that inactivate an enzyme involve mutant stop
codons.
Notes: Many genetic diseases are caused by point mutations that inactivate some
important enzyme or other protein. Very often, such inactivating mutations are
heterogeneous; deletions, insertions, frameshift mutations, and point mutations
may all give rise to the same clinical picture. The only group of patients that may
benefit from translational antitermination are those who carry a point mutation
that creates a premature stop codon in an otherwise intact open reading frame. In
this situation, it may be possible to restore expression of a functional protein by
promoting translational miscoding, that is, incorporation of some amino acid or
other vis-a-vis the mutant stop codon, which then enables the ribosome to keep
translating right through to the regular stop codon.1
1
If you are wondering now how this can be done without suppressing the orderly termination of
translation at the regular stop codon, I have no answer for you, but nevertheless commend you for
19.1 General considerations 357
Ribosomes Regular stop codon
Nascent proteins
Functional protein
Mutant stop codon
Incomplete protein
PTC124
Functional protein
Phase 1 Phase 2
10
Nasal potential difference (mV)
5
pathological
range
0
-5
-10
normal
range
-15
-20
0 14 28 42
Time (days)
paying attention. In case you were not wondering about this, you were probably not really studying
but just cramming for the exam.
Translational miscoding will not usually restore the original amino acid that was present before
the stop mutation occurred. Therefore, a further requirement is that the amino acid residue in
question is not critical for protein activity.
358 19 Enzyme and gene therapy of enzyme defects
Notes: The effectiveness of ataluren in various genetic diseases has been tested in
clinical pilot studies. This slide illustrates one such study, which was performed on
cystic fibrosis patients. This disease is caused by a deficiency in an ABC transporter
that exports chloride ions from cells, which impedes the secretion of fluid in all
kinds of exocrine glands. Multiple organs are functionally impaired, but the most
serious consequences arise from the buildup of viscous mucus in the lungs, which
facilitates chronic bacterial infections and ultimately leads to organ destruction.
In a sizable minority of all cystic fibrosis patients, the gene defect consists
in a premature stop codon, and in this group translational antitermination is
a plausible therapeutic strategy. The data illustrate the response to the drug
among such patients, during two successive phases of treatment. The effect of
the drug was measured as the electric potential difference that exists across the
nasal mucous membrane; this potential is increased when the chloride export by
the epithelial cells is reduced. Lines connect measurements on individual patients
before and after each course of treatment. Evidently, most patients experienced
a reduction in potential, that is, an increase in chloride export. Figure prepared
from original data in [133].
within the genome cannot be reliably controlled. Integration may occur in the vicin-
ity of some cancer-related gene, causing its transcriptional up- or downregulation.
Cases of leukemia have been induced in experiments both in humans and animals.
Adenoviruses are double-stranded DNA viruses that replicate without inte-
gration into the host cell genome. Accordingly, adenovirus-derived vectors will
not insert into the genome, which is safer. However, such episomal replication
will typically not be as long-lasting and stable as that of the retroviral vectors. If
repeated application of the vector is required, this will likely work only a limited
number of times, since, like the wild-type viruses, the coated vector particles are
immunogenic, and antibodies will interfere with their entry into cells.
Viruses of the Herpes family remain episomal yet are persistent, which is in
principle a very favorable combination. While their very large genome—Herpes
viruses are second only to Pox viruses in this regard—makes their use as vectors
technically challenging, they might well emerge as the vectors of choice in the long
run. An overview of the state of the art concerning viral vectors can be found in
[134].
19.2 Explain the advantages and disadvantages of retroviral vectors for gene therapy.
Adenosine deaminase (ADA) catalyzes the first step in the degradation of adeno-
sine and deoxyadenosine (see section 16.5). Deoxyadenosine accumulates and is
phosphorylated by salvage kinases, which yields dATP. Accumulating dATP exer-
cises feedback inhibition on ribonucleotide reductase [135], which is required in
the de novo synthesis of all deoxynucleotides (see slide 16.8).
Ribonucleotide reductase dI
Adenosine
deaminase
The apoptotic demise of the T-cells becomes manifest in early infancy in the
form of a severe combined immunodeficiency (SCID). The deficiency is referred to
as combined because it affects both humoral immunity, that is, antibody formation,
and cellular immunity, which is mediated by killer T-cells. Without treatment, the
children die after a few years; aggressive treatment is therefore justified.
NH2 NH2 HO H
N N N
N N
NH
HO
O
N N ! O
N N HO
O
N
N
OH OH OH OH
2
An identical twin would not be helpful with ADA deficiency, because he or she would suffer from
the same enzyme defect. However, identical twins come in handy with bone marrow transplants in
leukemia; the same goes for other organ transplants. Therefore, I urge you to get yourself a twin at
the earliest opportunity.
19.2 Adenosine deaminase deficiency 361
affects the 5-hydroxy group of the sugar, it is pretty obvious why this molecule is
not a substrate of the kinase.
This inhibitor is effective in principle but not sufficient. There is a second
enzyme, which is named deoxycytidine kinase but actually has a broader specificity
than suggested by this name, and which also phosphorylates deoxyadenosine. In
studies on cell cultures, this enzyme had to be inhibited also in order to permit
lymphocytes to survive [137]. This inhibitory approach has so far only been tried
in vitro; I have not yet seen any follow-up studies on its suitability in vivo.
Pentostatin is an inhibitor of adenosine deaminase. This obviously isn’t useful
in treating ADA deficiency, but it can be used to create experimental animal models
of the condition. It is clinically used to kill lymphocytes in graft-versus-host
reactions, as well as in certain forms of lymphocyte-derived malignancies.
19.3 Could 5-deoxyadenosine cause cytotoxicity by being incorporated into DNA? Why
not?
Searching PubMed for the terms in the title led me to this gem: Adenosine Deami-
nase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation
in Priapism [138]
Notes: Priapism is a state of sustained erection without sexual arousal and is
reportedly quite painful. It seems that an increased level of extracellular adenosine,
which activates adenosine receptors, can induce priapism. One possible source of
extracellular adenosine is the decay of red blood cells, for example in sickle cell
anemia3
According to the study cited, application of adenosine deaminase enzyme is
an effective treatment for priapism. Interesting, for sure, but it has of course
nothing to do with adenosine deaminase deficiency, other than that the enzyme
was readily available for this therapeutic experiment because of its common use
in the treatment of ADA deficiency.
Another interesting find was this early study: Enzyme replacement therapy for
adenosine deaminase deficiency and severe combined immunodeficiency
• strategy: application of frozen irradiated red blood cells (!)
• therapy improved immune status and helped patient survive for 17 months
(while waiting for blood marrow transplant)
Notes: This study [140] was performed before the advent of recombinant methods
for enzyme expression and purification. The use of red cells as carriers of the
enzyme is really quite ingenious! Adenosine and deoxyadenosine can leave and
3
One might expect priapism to occur in hemolytic crises in glucose-6-phosphate dehydrogenase
deficiency also; anecdotal evidence seems to support this assumption [139]).
362 19 Enzyme and gene therapy of enzyme defects
enter cells through nucleoside transporters (which have been discussed before,
see slides 16.4.1 and 16.9.8), and can therefore undergo degradation inside the
transfused red cells.
Notwithstanding the ingenuity of this treatment, it is bound to result in iron
overload in the long term (see slide 17.5.3). A better form of treatment is to use
the purified adenosine deaminase enzyme instead of blood transfusions. With
modern means, the recombinantly expressed human enzyme would seem to be
the obvious choice. At least in those patients that still express a nonfunctional
version of the enzyme—as opposed to no enzyme at all; both variants occur—
this enzyme should have the lowest likelihood of inducing inactivating antibodies.
However, it seems that currently a bovine enzyme preparation is still in use [141].
Presumably, ADA deficiency patients are more tolerant toward the application of a
non-self protein because of their immune defect. Furthermore, the bovine enzyme
is modified with PEG, both to reduce its immunogenicity and to increase its dwell
time in the system.4
While the inherent immunosuppression of ADA patients will facilitate the
use of purified enzyme, it will also make the patients more susceptible to graft-
versus-host reactions, that is, the proliferation of and immunological aggression by
transferred lymphocytes. In the study cited [140], the blood cells were irradiated
before transfusion, presumably in order to destroy any remaining lymphocytes
and thus prevent this complication.
19.4 Why can transfusion of red blood cells be used for enzyme therapy of adenosine
deaminase deficiency?
Still at the stage of clinical studies, not mainstream. A recent study [143] was
performed as follows:
• Non-myeloablative conditioning
• CD34+ bone marrow cells (stem cells) were isolated from the blood, trans-
duced in vitro with a retroviral vector carrying a functional ADA gene, and
reintroduced into the body
• ADA expression achieved in lymphocytes: ~5% in bone marrow, ~75% in pe-
riphery
• All patients survived at time point of compilation of study (2–8 years after
treatment), but some required additional enzyme treatment
Notes: The non-myeloablative conditioning used in this study means that the pa-
tients’ bone marrow was not entirely destroyed after harvesting of stem cells.
Therefore, after treatment, the genetically modified cells co-existed with non-
modified ones.5 It is noteworthy that a full 75% of T-lymphocytes in the peripheral
4
A good review on the use of PEG to modify enzymes and other therapeutic proteins is [142].
5
Myeloablative conditioning is the norm in allogenic bone marrow transplants, which is most
commonly performed as the treatment of last resort in leukemia or lymphoma patients.
19.3 Enzyme therapy of lysosomal enzyme defects 363
blood expressed ADA, while only 5% of the cells in the bone marrow did. This
discrepancy is most likely due to the survival advantage of the ADA-expressing
T-cells.
My interpretation of this cautious approach is that the investigators wanted to
keep the option of an allogenic bone marrow transplant, in case the gene therapy
failed. Myeloablative conditioning is very aggressive, and it can be used only
once on each patient. Considering the fairly favorable outcome of this cautious
initial experiment, it seems to me that a somewhat more aggressive approach,
with a larger number of stem cells being transformed, and with a more intense
decimation of the remaining bone marrow in the conditioning phase, is justified.
19.5 Explain the various form of therapy that can be used to treat adenosine deaminase
deficiency.
Adenosine deaminase is active in the cytoplasm, and since the substrates can
enter and leave the cells by facilitated diffusion, there is no major problem with
arranging for the enzyme to meet its substrate. However, this is different in
lysosomal enzyme defects.
Lysosomes contain a diverse range of hydrolytic enzymes (hydrolases), which
are important in the breakdown of both extracellular and intracellular molecules.
These enzymes have an acidic pH optimum, which matches the acidic environment
inside phagolysosomes, that is, the vesicles that form by fusion of substrate-
containing vesicles with lysosomes.
There are quite a few hereditary enzyme defects that concern one or the other
lysosomal hydrolase, and depending on the type of compound that now can no
longer be degraded and therefore accumulates, these diseases are referred to as
lipidoses, mucopolysaccharidoses, or glycogenoses.
When a lysosomal enzyme is deficient, the substrate nevertheless continues
to be targeted to phagolysosomes. Therefore, in order to treat this condition with
enzyme therapy, we need to get the enzyme into the lysosomes as well. We will
look at some examples to understand how this can be achieved.
Notes: In Pompe disease, the deficient enzyme is acid maltase (see slide 8.6.2).
Without this enzyme, lysosomal glycogen degradation stalls (see slide 8.3.7), and
undegraded glycogen particles slowly accumulate. The accumulation of glycogen
concerns mostly skeletal and heart muscle and leads to the degeneration of these
tissues.
Notes: The two continuous curves at the top are the references for boys and girls.
The shorter graphs at the bottom show the data from individual patients over the
time course of the study. It is evident that muscle strength improves but remains
below normal.
Figure adapted from [144]. Also note the very clever color coding used in the
figure.
19.3 Enzyme therapy of lysosomal enzyme defects 365
Notes: This cartoon—loosely based on one from [145]—illustrates the role of the
mannose-6-phosphate receptor in targeting proteins to the lysosomes and endo-
somes. Proteins that contain the sugar mannose-6-phosphate in their glycosyl
moieties can bind to the mannose-6-phosphate receptor, a membrane protein that
occurs in several types of intracellular vesicles and compartments. The receptor
picks up lysosomal enzymes within the trans-Golgi network and ensures that they
indeed make their way to the lysosomes. Acid maltase is one of the proteins that
are transported to the lysosomes by this mechanism.
Some mannose-6-phosphate receptor molecules also make their way to the
cell surface. These receptor molecules can bind to acid maltase that was applied
therapeutically and is present in the extracellular space. The receptor-enzyme
complexes will undergo endocytosis and find themselves in endosomes that re-
versibly fuse with lysosomes. Hitching a ride inside a vagrant lysosome, an enzyme
molecule may eventually alight upon the undegraded substrate within another en-
dosome. Considering how roundabout and haphazard this transport process is, it
is perhaps not surprising that the targeting of therapeutically applied lysosomal
enzymes is not very efficient.
Notes: Protein glycosylation occurs within the Golgi apparatus and the trans-Golgi
network. It is a stepwise process, in which several different glycosyltransferases
attach sugar moieties to the ends of growing oligosaccharide chains. The composi-
tion of the oligosaccharide moieties attached to a given protein can vary with the
host cell that expresses it. In the case of lysosomal enzymes, such differences may
affect the rate of uptake into cells.
The experimental study summarized in this slide illustrates the importance
of the carbohydrate moiety for cellular uptake. Acid maltase was expressed in
366 19 Enzyme and gene therapy of enzyme defects
control CHO cells (blue lines) and in a derived cell line engineered for increased
incorporation of mannose-6-phosphate into the oligosaccharides (green lines, CHO
M6P ). The enzyme expressed in these cells contains greater amounts of mannose-
6-phosphate than a control sample obtained from CHO cells (left), and it is indeed
taken up more efficiently into cells lacking the enzyme (right). Figure prepared
from original data in [146].
CHO M6P
12
CHO wt 6
0
10 20 30 40 50
Man-6- P 2 Man-6- P Enzyme in medium (nM)
In the following slides, we will, as a final example, take a look at Gaucher dis-
ease. This is another lysosomal enzyme defect that displays some interesting and
instructive differences to Pompe disease.
HO O HO OH OH
H H
N N
HO O HO O
H2 O
glucosylceramide ceramide
NeuNAc
100
Gal Gal
Enzyme in plasma (%)
GlcNAc GlcNAc
Man Man
Man
GlcNAc 10
0 5 10 15 20 25 30
GlcNAc Fuc Time after injection (min)
Enzyme protein
6
Another aspect that likely facilitates the uptake of enzyme by macrophages is the absence
of an endothelial barrier. While muscle cells are separated from the blood flow by a continuous
endothelium, the macrophages in the spleen and liver reside directly within the blood-percolated
sinusoids.
368 19 Enzyme and gene therapy of enzyme defects
O O
OH
OH
HO OH
N NH
N O
OH
miglustat eliglustat
Notes: These two drugs are inhibitors of glucocerebroside synthesis. Total inhi-
bition of glucocerebroside synthesis is not compatible with life, so this strategy
does not obviate the need for enzyme therapy and is not free of side effects.
Serendipitously, miglustat was also found to inhibit spermatogenesis in mice,
suggesting a new approach for male contraception. This effect, however, could not
be substantiated in men [148].
19.6 Explain the fundamental problem with enzyme therapy of lysosomal enzyme defects,
and how it can be overcome. Explain how enzyme therapy works in Pompe disease and in
Gaucher disease.
3. Gene therapy: Intact copies of the adenosine deaminase gene are transferred into
the patient’s own bone marrow stem cells. Potentially curative if genes are stably ex-
pressed. Avoids immunological complications of bone marrow transplant but, at least
with retroviral vectors, has risks such as leukemia.
4. Drug therapy: Inhibition of salvage pathway kinases that convert deoxyadenosine
to dATP. Experimental and unproven.
19.6: The fundamental problem is that lysosomal enzymes are active only inside lyso-
somes, but intravenously applied enzymes are not usually transported into lysosomes.
The problem can be overcome by endowing the enzymes with oligosaccharide moieties
which are recognized by cellular receptors that trigger endocytosis.
On nucleated cells in general, endocytosis and transport to the lysosomes is facilitated
by the mannose-6-phosphate receptor. This is exploited in Pompe disease, which is caused
by a lack of the lysosomal enzyme acid maltase. Recombinantly expressed acid maltase
carrying mannose-6-phosphate is taken up by muscle cells via the mannose-6-phosphate
receptor.
Gaucher disease affects macrophages; the missing enzyme is glucocerebrosidase.
These cells have a mannose receptor, which normally serves to mediate phagocytosis of
microbes. Glucocerebrosidase is purified and partially deglycosylated in order to expose
mannose residues contained within its natural oligosaccharide moiety. This modified
enzyme is rapidly and efficiently taken up by macrophages.
Chapter 20
Most of the illustrations in these notes were created by me; this is the case for all
plots, reaction schemes, molecular structures, and cartoons, except where stated
otherwise. These images—like the text, which was entirely written by me—are
subject to the terms of the copyright notice below.
Photographs were obtained from various sources. Most of the histological
images were obtained from pathorama.ch and are reproduced here with the kind
permission of that website’s owner, Katharina Glatz. All micrographs for which
another source is not specified below were obtained from this source.
For all other photographs, the sources are listed below, as far as I have been
able to ascertain them. These images have been used either with explicit permis-
sion of the originating websites’ owners, or under the following rationales:
• Images from wikipedia: These are usually released under the Creative Com-
mons 3.0 license, which permits their incorporation, in unchanged form, into
a collection, without affecting the copyright of the resulting collection as a
whole. I believe that these lecture notes and slides constitute a collection
conforming to this clause.
• Images from individual sources, for which no copyright holder could be iden-
tified, or for which requests for permission to reuse went unanswered: the
use of these images is deemed appropriate (1) because of general fair use
considerations, and/or (2) because the images in question constitute faithful
reproductions of physical phenomena, without artistic originality, and there-
fore, according to a decision by the US Court of Appeals, are not subject to
copyright protection.
If you have the rights to an image used without explicit permission and credit,
and you do not agree with its use in these notes, please let me know, and I will
371
372 20 Credits and copyright
remove that image. If you have the rights and you do agree with its use, I would
appreciate a brief note to this effect, so that I can give you the proper credit.
Slide Source
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374
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