INTERNATIONAL UNIVERSITY

SCHOOL OF BIOTECHNOLOGY

CHAPTER 2: CARBONHYDRATE
METABOLISM- GLYCOLYTIC ENZYMES

BIOCHEMISTRY

1
 Learning objectives

1. Review the carbonhydrates

2. Learn the names of the 10 enzymes of glycolysis.
3. Learn the structures of the intermediates in the
glycolytic pathway.
4. Explore the structures of the glycolytic enzymes.
5. Understand the chemical mechanisms of the enzymes
of glycolysis.

2
 Brief Content
1.The carbonhydrate
2.The glycolytic enzymes
2.1 Hexokinase
2.2 Phosphoglucose Isomerase
2.3 Phosphofructokinase
2.4 Aldolase
2.5 Triose phosphate Isomerase
2.6 Glyceraldehide-3- phosphate dehydrogenase
2.7 Phosphoglycerate kinase
2.8 Phosphoglycerate mutase
2.9 Enolase
2.10 Pyruvate kinase

3
 Detailed Content
1.The carbonhydrate
2.The glycolytic enzymes
2.1 Hexokinase
Structure
Catalytic mechanism
Active site details
2.2 Phosphoglucose Isomerase
Structure
Catalytic mechanism
4 Active site details
 Detailed Content
2.3 Phosphofructokinase
Structure
Catalytic mechanism
Active site details
2.4 Aldolase
Structure
Catalytic mechanism
Active site details

5
 Detailed Content
2.5 Triose phosphosphate Isomerase
Structure
Catalytic mechanism
Active site details
2.6 Glycealdehide-3- phosphate dehydrogenase
Structure
Catalytic mechanism
Active site details

6
 Detailed Content
2.7 Phosphoglycerate kinase
Structure
Catalytic mechanism
Active site details
2.8 Phosphoglycerate mutase
Structure
Catalytic mechanism
Active site details

7
 Detailed Content
2.9 Enolase
Structure
Catalytic mechanism
Active site details
2.10 Pyruvate kinase
Structure
Catalytic mechanism
Active site details

8
 * Sugars, the smallest carbohydrates, serve as
fuel and carbon sources
1 CARBOHYDRATES: * Polysaccharides, the polymers of sugars,
have storage and structural roles

Carbohydrates include both sugars and

their polymers. The simplest
carbohydrates are the monosaccharides,
or single sugars, also known as simple
sugars. Disaccharides are double sugars,
consisting of two monosaccharides joined
by condensation. The carbohydrates that
are macromolecules are polysaccharides,
polymers of many sugars.
9
 1.1. Sugars, the smallest carbohydrates, serve as fuel and carbon sources

- Monosaccharides (from the Greek monos, single, and sacchar, sugar) generally
have molecular formulas that are some multiple of CH 2O.
- Glucose (C6H12O6), the most common monosaccharide, is of central importance in
the chemistry of life. In the structure of glucose, we can see the trademarks of a sugar:
- The molecule has a carbonyl group and multiple hydroxyl groups. Depending on the
location of the carbonyl group, a sugar is either an aldose (aldehyde sugar) or a ketose
(ketone sugar).
- Glucose, for example, is an aldose; fructose, a structural isomer of glucose, is a
ketose. (Most names for sugars end in -ose. )

- Monosaccharides, particularly glucose, are major nutrients for cells. In the process
known as cellular respiration, cells extract the energy stored in glucose molecules.
- Not only are simple sugar molecules a major fuel for cellular work, but their carbon
skeletons serve as raw material for the synthesis of other types of small organic
molecules, such as amino acids and fatty acids.
10 - Sugar molecules that are not immediately used in these ways are generally
 Fig 3.1. The structure and classification of some monosaccharides.
Sugars may be aldoses (aldehyde sugars) or ketoses (ketone sugars), depending on the location of
the carbonyl group (pink). Sugars are also classified according to the length of their carbon
skeletons. A third point of variation is the spatial arrangement around asymmetric carbons
(compare, for example, the gray portions of glucose and galactose).
11
 Fig 3.2. Linear and ring forms of glucose
12
 - A disaccharide consists of two monosaccharides joined by a
glycosidic linkage, a covalent bond formed between two
monosaccharides by a dehydration reaction. For example, maltose is a
disaccharide formed by the linking of two molecules of glucose.
- Also known as malt sugar, maltose is an ingredient for brewing beer.
The most prevalent disaccharide is sucrose, which is table sugar. Its
two monomers are glucose and fructose.
- Plants generally transport carbohydrates from leaves to roots and
other nonphotosynthetic organs in the form of sucrose.
- Lactose, the sugar present in milk, is another disaccharide, consisting
of a glucose molecule joined to a galactose molecule.

13
 Fig 3.3.
Examples
of
disaccharide
synthesis

14
 1.2 Polysaccharides, the polymers of sugars,
have storage and structural roles

- Polysaccharides are macromolecules, polymers with a

few hundred to a few thousand monosaccharides joined
by glycosidic linkages.
- Some polysaccharides serve as storage material,
hydrolyzed as needed to provide sugar for cells.
- Other polysaccharides serve as building material for
structures that protect the cell or the whole organism.
- The architecture and function of a polysaccharide are
determined by its sugar monomers and by the positions of
its glycosidic linkages.
15
 1.2.1 Storage polysaccharides

- Starch, a storage polysaccharide of plants, is a polymer consisting entirely of

glucose monomers. Most of these monomers are joined by 1-4 linkages (number 1
carbon to number 4 carbon), like the glucose units in maltose. The angle of these
bonds makes the polymer helical.
- The simplest form of starch, amylose, is unbranched. Amylopectin, a more
complex form of starch, is a branched polymer with 1-6 linkages at the branch
points.
- Plants store starch as granules within cellular structures called plastids, including
chloroplasts (see fig5-6a). By synthesizing starch, the plant can stockpile surplus
glucose. Because glucose is a major cellular fuel, starch represents stored energy.
The sugar can later be withdrawn from this carbohydrate bank by hydrolysis, which
breaks the bonds between the glucose monomers.
- Animals store a polysaccharide called glycogen, a polymer of glucose that is like
amylopectin but more extensively branched. Humans and other vertebrates store
glycogen mainly in liver and muscle cells. Hydrolysis of glycogen in these cells
16 releases glucose when the demand for sugar increases.
 Fig 3.4. Storage polysaccharides.
These examples, starch and glycogen, are composed entirely of glucose monomers,
abbreviated here as hexagons. The polymer chains tend to spiral to form helices.
17
 1.2.2. Structural polysaccharides

- Organisms build strong materials from structural polysaccharides. For

example, the polysaccharide called cellulose is a major component of the
tough walls that enclose plant cells. On a global scale, plants produce almost
1011 (100 billion) tons of cellulose per year; it is the most abundant organic
compound on Earth.
- Like starch, cellulose is a polymer of glucose, but the glycosidic linkages in
these two polymers differ. The difference is based on the fact that there are
actually two slightly different ring structures for glucose.
- When glucose forms a ring, the hydroxyl group attached to the number 1
carbon is locked into one of two alternative positions: either below or above the
plane of the ring. These two ring forms for glucose are called alpha (a) and beta
(b), respectively.
- In starch, all the glucose monomers are in the a configuration.
- In contrast, the glucose monomers of cellulose are all in the b configuration,
making every other glucose monomer upside down with respect to the others.
18
 Fig 3.5.
Starch and
cellulose
structures

19
 - The differing glycosidic links in starch and cellulose give the two
molecules distinct three-dimensional shapes. Whereas a starch
molecule is mostly helical, a cellulose molecule is straight (and never
branched), and its hydroxyl groups are free to hydrogen-bond with the
hydroxyls of other cellulose molecules lying parallel to it.
- In plant cell walls, parallel cellulose molecules held together in this
way are grouped into units called microfibrils. These cables are a
strong building material for plants--as well as for humans, who use
wood, which is rich in cellulose, for lumber.

- Enzymes that digest starch by hydrolyzing its a linkages are unable

to hydrolyze the b linkages of cellulose. In fact, few organisms
possess enzymes that can digest cellulose. Humans do not; the
cellulose fibrils in our food pass through the digestive tract and are
20 eliminated with the feces.
 Some microbes can digest cellulose, breaking it down to glucose
monomers. Another important structural polysaccharide is chitin,
the carbohydrate used by arthropods (insects, spiders,
crustaceans, and related animals) to build their exoskeletons. An
exoskeleton is a hard case that surrounds the soft parts of an
animal. Pure chitin is leathery, but it becomes hardened when
encrusted with calcium carbonate, a salt.

- Chitin is also found in many fungi, which use this polysaccharide

rather than cellulose as the building material for their cell walls.

- Chitin is similar to cellulose, except that the glucose monomer of

chitin has a nitrogen-containing appendage:

21
 Fig 3.6. The arrangement of cellulose in plant cell walls.
22
 Fig 3.7. Chitin, a structural polysaccharide.
(a) Chitin forms the exoskeleton of arthropods. This
cicada is molting, shedding its old exoskeleton and
emerging in adult form.
(b) Chitin is used to make a strong and flexible
surgical thread that decomposes after the wound
or incision heals

23
 THE STRUCTURE OF ATP, NAD+

- The molecule known as ATP, short for adenosine triphosphate, is the central character
in bioenergetics

- The triphosphate tail of ATP is the chemical equivalent of a loaded spring; the close
packing of the three negatively charged phosphate groups is an unstable, energy-storing
arrangement. The chemical "spring" tends to "relax" by losing the terminal phosphate

- The cell taps this energy source by using enzymes to transfer phosphate groups from
ATP to other compounds, which are then said to be phosphorylated. Phosphorylation
primes a molecule to undergo some kind of change that performs work, and the molecule
loses its phosphate group in the process
ATP = ADP + Pi

- For example, a working muscle cell, for example, recycles its ATP at a rate of about 10
million molecules per second. To understand how cellular respiration regenerates ATP,
24 we need to examine the fundamental chemical processes known as oxidation and
reduction.
 Fig 3.8. The
structure and
hydrolysis of ATP.
The hydrolysis of ATP
yields inorganic
phosphate and ADP.
In the cell, most
hydroxyl groups of
phosphates are
ionized (--O-).

25
 Fig 3.9. NAD+ as an electron shuttle.
The full name for NAD+, nicotinamide adenine dinucleotide, describes its structure;
the molecule consists of two nucleotides joined together. (Nicotinamide is a
nitrogenous base, although not one that is present in DNA or RNA.) The enzymatic
transfer of two electrons and one proton from some organic substrate to NAD+
reduces the NAD+ to NADH. Most of the electrons removed from food are
transferred initially to NAD+.
26
 - Electrons lose very little of their potential energy when they are
transferred from food to NAD+.
- Each NADH molecule formed during respiration represents stored
energy that can be tapped to make ATP when the electrons complete their
"fall" from NADH to oxygen

- How do electrons extracted from food and stored by NADH finally reach
O2?
(1) The reaction between H2 and O2 to form H2O + gases = explosion +
release of energy
(2) Cellular respiration also brings H2 and O2 together to form H2O, but there
are two important differences. First, in cellular respiration, the H2 that reacts
with O2 is derived from organic molecules. Second, respiration uses an
electron transport chain to break the fall of electrons to O2 into several
27 energy-releasing steps instead of one explosive reaction
 - The transport chain consists of a number of molecules, mostly
proteins, built into the inner membrane of a mitochondrion.
- Electrons removed from food are shuttled by NADH to the
"top" end of the chain. At the "bottom" end, O2 captures
these electrons along with H2, forming water.

- Thus, electrons removed from food by NAD+ fall down the

electron transport chain to a far more stable location in the
electronegative O2 atom.

Food  NADH  electron transport chain 8n oxygen

28
 Fig 3.10. An introduction to electron transport chains.
(a) The uncontrolled exergonic reaction of H2 with O2 to form H2O releases a large
amount of energy in the form of heat and light: an explosion.
(b) In cellular respiration, the same reaction occurs in stages: An electron transport
chain breaks the "fall" of electrons in this reaction into a series of smaller steps and
stores some of the released energy in a form that can be used to make ATP.
29 (The rest of the energy is released as heat.)
 THE PROCESS OF CELLULAR RESPIRATION

* Respiration involves glycolysis, the Krebs cycle,

and electron transport:
* Glycolysis harvests chemical energy by oxidizing
glucose to pyruvate:
* The Krebs cycle completes the energy-yielding
oxidation of organic molecules:
* The inner mitochondrial membrane couples
electron transport to ATP synthesis:
* Cellular respiration generates many ATP molecules
for each sugar molecule it oxidizes:
30
 Respiration involves glycolysis, the Krebs cycle,
and electron transport

- In a eukaryotic cell, glycolysis occurs outside the mitochondria in

the cytosol.
The Krebs cycle and the electron transport chains are located
inside the mitochondria.
- During glycolysis, each glucose molecule is broken down into
two molecules of the compound pyruvate.
- The pyruvate crosses the double membrane of the
mitochondrion to enter the matrix, where the Krebs cycle
decomposes it to carbon dioxide.
- NADH or FADH2 transfers electrons from molecules undergoing
glycolysis and the Krebs cycle to electron transport chains,
which are built into the inner mitochondrial membrane.
31
 The electron transport chain converts the chemical energy to a
form that can be used to drive oxidative phosphorylation, which
accounts for most of the ATP generated by cellular respiration.
- A smaller amount of ATP is formed directly during glycolysis and
the Krebs cycle by substrate-level phosphorylation.

(1) Glycolysis (color-coded teal throughout the chapter)

(2) The Krebs cycle (color-coded salmon)
(3) The electron transport chain and oxidative phosphorylation
(color-coded violet)

32
33 Fig.3.11: Overview of the cellular respiration
 Respiration is a cumulative function of 3 metabolic stages
(1) Glycolysis (color-coded teal throughout the chapter) - cytosol
Glycolysis, begins the degradation by breaking: glucose = two molecules pyruvate

(2) The Krebs cycle (color-coded salmon) - mitochondrial matrix

Decomposing a derivative of pyruvate to CO2

(3) The electron transport chain and oxidative phosphorylation (color-coded violet)
the electron transport chain accepts electrons from the breakdown products of the first
two stages (usually via NADH) and passes these electrons from one molecule to
another

34
  The energy released at each step of the chain is stored in a form the
mitochondrion can use to make ATP.
 - This mode of ATP synthesis is called oxidative phosphorylation because it
is powered by the redox reactions that transfer electrons from food to O2.

 - Oxidative phosphorylation accounts for almost 90% of the ATP generated by

respiration
 - A smaller amount of ATP is formed by substrate-level phosphorylation
when an enzyme transfers a phosphate group from a substrate molecule to
ADP. "Substrate molecule" here refers to an organic molecule generated
during the catabolism of glucose.

 cell respiration
 glucose = CO2 + H2O + 38 molecules of ATP

35
 Fig 3.12. Substrate-level phosphorylation.
Some ATP is made by direct enzymatic transfer of a phosphate group from a substrate to
36 ADP. The phosphate donor in this case is phosphoenolpyruvate (PEP), which is formed
from the breakdown of sugar during glycolysis
 2. INTRODUCTION

Glycolysis is an almost universal pathway for extraction of the

energy available from carbohydrates, shared among prokaryotes
and eukaryotes, aerobes and anaerobes alike. In anaerobes,
glycolysis is the only significant source of energy from
carbohydrates. In aerobic organisms, considerably more energy
can be harvested downstream from glycolysis in the citric acid
cycle. Glycolysis produces energy in the form of ATP and NADH.

The glycolytic pathway consists of 10 enzyme-catalyzed steps.

During glycolysis, glucose, a six-carbon carbohydrate, is oxidized
to form two molecules of pyruvate, a three-carbon molecule. For
each glucose molecule metabolized, the pathway produces two
molecules of ATP and two molecules of NADH.

37
 2. INTRODUCTION

Glycolysis is not isolated from other metabolic pathways. Other

molecules besides glucose can enter at a few points along the
glycolytic pathway. For example, the product of glycogen
breakdown glucose-6-phosphate, can enter the glycolytic
pathway at the second step. Glyceraldehyde-3-phosphate,
which is produced by photosynthesis, is also a glycolytic
intermediate, so it can be directed from this anabolic pathway
into glycolysis when energy is needed. Additionally,
intermediates can be drawn out of the glycolytic pathway when
energy levels are high, for use in biosynthetic pathways. For
instance, during active energy production pyruvate, the product
of glycolysis, enters the citric acid cycle, but when energy is not
needed pyruvate serves as a substrate in amino acid synthesis.

38
 Fig 3.13: Reactions
of Glycolysis

39
 2. GLYCOLYSIS
Glycolysis harvests chemical energy by oxidizing glucose to pyruvate

- Glucose,a six-carbon sugar, is split into two three-carbon sugars.

These smaller sugars are then oxidized and their remaining atoms
rearranged to form two molecules of pyruvate. (Pyruvate is the ionized form
of a three-carbon acid, pyruvic acid.)

- The pathway of glycolysis consists of ten steps, each catalyzed by a

specific enzyme. We can divide these ten steps into two phases: The
energy investment phase includes the first five steps, and the energy
payoff phase includes the next five steps.
- During the energy investment phase, the cell actually spends ATP to
phosphorylate the fuel molecules and NAD+ is reduced to NADH by
oxidation of the food
glycolysis
40 glucose = 2 ATP + 2 NADH
 Fig 3.14. The energy input and output of glycolysis
41
 Fig 3.15 . A closer look at glycolysis.
The orientation diagram at the right relates glycolysis to the whole process of respiration.
Do not let the chemical detail in the main diagram block your view of glycolysis as a
source of ATP and NADH.
42
 Fig 3.16. A closer look at glycolysis.
The orientation diagram at the right relates glycolysis to the whole process of respiration.
Do not let the chemical detail in the main diagram block your view of glycolysis as a
source of ATP and NADH.
43
 2. 1. Hexokinase

PHOSPHORYLATION OF GLUCOSE

The first glycolytic reaction attaches a phosphate

group to glucose, to yield glucose-6-phosphate. This
reaction is catalyzed by the enzyme hexokinase,
shown here with its substrates bound in the active
site. A kinase is an enzyme that catalyzes the
transfer of a phosphoryl group to or from ATP. In this
case, the phosphoryl group is transferred from ATP
to glucose (thereby converting ATP to ADP).

44
 2. 1. Hexokinase

Structure

Hexokinase is a homodimer. Each subunit is made

of two globular domains linked by an α helix. The
subdomains are composed of two segments of β
sheet that are protected from the solvent by α
helices. The active sites are sandwiched between
the β sheets. The active site has space to bind both
glucose and ATP.

45
 3.17 The structure of hexokinase

46
 2.1. Hexokinase

Catalytic mechanism
The oxygen on carbon 6 of glucose performs α nucleophilic
attack on the γ phosphate of ATP. The phosphate electron pair
is donated to the anhydride oxygen bridging the β and γ
phosphates of the ATP. Thus, the glucose obtains α phosphate
from ATP.

Active site details

Glucose, the substrate of hexokinase, is cradled snugly in the
enzyme's active site. It forms hydrogen bonds with amino acid
side chains and the protein backbone.

47
 3.18 Catalytic mechanism of
hexokinase

48
 Fig. 3.19: Active
site of
hexokinase

49
 2. 2. Phosphoglucose Isomerase

ISOMERIZATION OF GLUCOSE-6-PHOSPHATE TO
FRUCTOSE-6-PHOSPHATE

The second reaction is the isomerization of glucose-

6-phosphate to fructose-6-phosphate by the enzyme
phosphoglucose isomerase. Isomers have the same
chemical formula (being composed of the same
atoms), but with a different arrangement of bonds.

50
 2. 2. Phosphoglucose Isomerase

Structure

Phosphoglucose isomerase is a homodimer composed

of two globular subunits that embrace each other with
flanking a helical "arms." Each subunit is composed of
one mixed and one parallel β sheet. These are
separated by a central bundle of α helices. The active
sites are located symmetrically at the interface between
the two subunits near the flanking "arms."

51
 Fig 3.20:
Structure of
Phosphoglucose
isomerase

52
 2.2 Phosphoglucose Isomerase

Catalytic mechanism

The mechanism of isomerization cannot proceed without first

opening the ring of glucose-6-phosphate. An active site acid
catalyzes the ring opening. A basic group then a abstracts the
proton attached to carbon 2 on the glucose. This leads to double
bond formation between carbon 1 and carbon 2 and an electron
displacement at the carbonyl of carbon 1. In the subsequent step, a
catalytic base abstracts a proton from the carbon 2 hydroxyl group,
leaving an unpaired electron that proceeds to form a carbonyl bond
with carbon 2. The remaining unpaired electron at carbon 1 then
abstracts a proton from an active site acid and the isomerization is
complete. Finally, ring closure produces the cyclic form of fructose-
6-phosphate that is free to leave the active site.
53
 Fig 3.21: Catalytic
mechanism of
phosphoglucose
Isomerase

54
 2.2 Phosphoglucose Isomerase

Active site details

The active site of phosphoglucose isomerase

contains two amino acids believed to be
directly involved in catalysis: a lysine acting
as an acid and a conserved glutamate that
functions as a base. Shown here is the active
site with bound product fructose-6-
phosphate.
55
 Fig. 3.22:Active site of phosphoglucose isomerase
56 concluding lysine, histidine, glutamate
 2.3. Phospho- fructokinase

PHOSPHORYLATION OF FRUCTOSE-6-PHOSPHATE TO
FRUCTOSE-1,6-BISPHOSPHATE

The third reaction in the glycolytic pathway is the

phosphorylation of the fructose-6-phosphate by the enzyme
phosphofructokinase (PFK) to produce fructose-1,6-
bisphosphate. This step requires an ATP, and because of the
highly favorable energetics of this irreversible reaction, it is
known as the committed step of glycolysis and is highly
regulated. Regulatory molecules include high-energy metabolic
intermediates, such as phosphoenolpyruvate (PEP), that inhibit
the activity of PFK, and low-energy intermediates, such as
adenosine diphosphate (ADP), that activate PFK. Both
activating and inhibitory effectors bind in the same binding
pocket located between the subunits of each dimer of PFK.
57
 2.3. Phospho- fructokinase

Structure

PFK exists as a tetramer in solution. Four identical

subunits associate to form the active form of PFK.
Like hemoglobin, PFK is a dimer of dimers. Each
subunit in the tetramer contains 319 amino acids that
form two domains. The larger domain binds the
substrate ATP, and the smaller domain binds the
other substrate, fructose-6-phosphate. Each domain
is a β barrel, consisting of a cylindrical β sheet
58 surrounded by a helices.
 Fig. 3.23:
Structure of
Phospho-
fructokinase

59
 2.3. Phospho- fructokinase

Catalytic mechanism

The catalytic mechanism of

phosphofructokinase is nearly identical to the
phosphorylation of glucose performed by
hexokinase. The hydroxyl oxygen of carbon
1 performs a nucleophilic attack on the β
phosphate of ATP. These electrons are
donated to the anhydride oxygen bridging the
β and γ phosphates of the ATP.
60
 Fig. 3.24: Catalytic
mechanism of
phosphofructokinase

61
 2. 3. Phospho- fructokinase

Active site details

The active site is located at the interface

between subunits. In this crystal structure of
PFK, ADP and glucose-6-phosphate are
bound to the active site.

62
 Fig. 3. 25: Active site of Phospho- fructokinase including lysine
and histidine
63
 2. 3. Phospho- fructokinase

Allosteric effector site

The allosteric effector site is located on the

opposite side of each subunit from the active
site, at the interface between subunits in the
dimer. This structure has ADP bound at this
site.

64
 Fig. 3.26: Allosteric
effector site of
Phospho- fructokinase

65
 2. 4. Aldolase

CLEAVAGE OF FRUCTOSE-1,6-BISPHOSPHATE INTO

DIHYDROXYACETONE PHOSPHATE AND
GLYCERALDEHYDE-3- PHOSPHATE

In the fourth reaction, the enzyme aldolase

cleaves the hexose fructose-1,6-
bisphosphate into two three-carbon
molecules, glyceraldehyde-3-phosphate and
dihydroxyacetone phosphate.

66
 2. 4. Aldolase

Structure
Aldolase is a tetramer of identical subunits.
Each subunit is composed of α β barrel
surrounded by β helices. The active site is
located at one end of the β barrel and can be
identified by the bound product
dihydroxyacetone phosphate.

67
 Fig. 3.27: Structure of
aldolase

68
 2. 4. Aldolase

Catalytic mechanism

The mechanism of aldolase involves the formation of a Schiff base,

a common covalent intermediate. In the first step, an active-site
lysine donates an electron to the carbon 2 carbonyl of fructose-1,6-
bisphosphate. Subsequent loss of water leads to the formation of
the Schiff base. A tyrosine anion abstracts a proton from the
carbon 4 hydroxyl group, leaving a carbonyl group. The displaced
electrons at carbon 3 form an enolate intermediate with carbon 2.
The displaced electron of the Schiff base now relocates to the
nitrogen. At this point, glyceraldehyde-3-phosphate has been
created and is free to diffuse out of the active site.

69
 2. 4. Aldolase

In subsequent steps, the Schiff base is

reestablished, and the enolate intermediate is
destroyed when the protonated tyrosine
donates its proton to the displaced electron of
carbon 3. Finally, a hydration step transforms
the Schiff base back to a carbonyl, and the
dihydroxyacetone phosphate product diffuses
out of the active site.

70
 Fig. 3.28: Catalytic
mechanism of aldolase

71
 2. 4. Aldolase

Active site details

Here you see the active site of aldolase

complexed with glyceraldehyde-3-phosphate
There is a histidine in the active site as well
as several lysines that might be the Schiff-
base forming residue.

72
 Fig. 3.29:
Active site
details of
aldolase

73
 2. 5. Triose Phosphate Isomerase

ISOMERIZATION OF DIHYDROXYACETONE PHOSPHATE TO

GLYCERALDEHYDE- 3-PHOSPHATE

The fifth reaction in the glycolytic pathway is the

isomerization of dihydroxyacetone phosphate to
glyceraldehyde-3-phosphate. Triose phosphate
isomerase is an example of a "perfect" enzyme or an
enzyme in which the rate of catalysis is limited only
by the ability of the enzyme to encounter its
substrate, rather than any chemical event.

74
 2. 5. Triose Phosphate Isomerase

Structure
Triose phosphate isomerase is a dimer made
from two globular subunits. Each subunit is
composed of a central β barrel surrounded
by α helices. In this respect, the secondary
structure is similar to the previous enzyme in
the pathway, aldolase.

75
76 Fig. 3.30: Structure of Triose Phosphate Isomerase
 2.5. Triose Phosphate Isomerase

Catalytic mechanism

In the first step of the mechanism, an acid donates a proton to

the carbonyl at carbon 2 of dihydroxyacetone phosphate. At the
same time, a basic group abstracts a proton at carbon 1. This
concerted action leads to the formation of an enediol
intermediate. Subsequently, the deprotonated acid group
accepts a proton from the hydroxyl group at carbon 1, as the
protonated basic group donates its proton to carbon 2. This
generates the glyceraldehyde-3-phosphate product. A
glutamate and a histidine act as the general acids and bases
for this isomerization reaction, and a lysine serves to stabilize
the negative charge of the transition state intermediate.

77
 Fig. 3.31: Catalytic
mechanism of Triose
Phosphate Isomerase

78
 2.5. Triose Phosphate Isomerase

Active site details

The structure of triose phosphate isomerase
can be seen here with glycerol-3-phosphate,
an analog of glyceraldehyde-3-phosphate.

79
80 Fig. 3.32: Active site details of Triose Phosphate Isomerase
 2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase

OXIDATION AND PHOSPHORYLATION OF

GLYCERALDEHYDE-3- PHOSPHATE

In the sixth reaction of glycolysis, glyceraldehyde-3-phosphate

is both oxidized and phosphorylated to produce 1,3-
bisphosphoglycerate. Unlike the kinases that catalyzed
reactions 1 and 3, glyceraldehyde-3-phosphate dehydrogenase
does not use ATP as a phosphoryl-group donor; it instead adds
inorganic phosphate directly to the substrate. This reaction is
also an oxidation-reduction reaction in which the aldehyde
group of glyceraldehyde-3-phosphate is oxidized with the
concomitant reduction of the cofactor NAD+ to NADH.

81
 2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase

Recent evidence supports a wide variety of

cellular roles for this enzyme beyond its
glycolytic function, including endocytosis,
translational control, tRNA export from the
nucleus, and DNA repair. Enzymes involved
in more than one cellular process often serve
as important regulators, connecting multiple
enzymatic pathways.

82
 2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase

Structure

Glyceraldehyde-3-phosphate dehydrogenase
is a tetramer. Each identical subunit is
composed of two extended β sheets, both
supported by three α helices.

83
 Fig. 3.33:
Structure of
Glyceraldehyde
- 3-phosphate
Dehydrogenase

84
 2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase

Catalytic mechanism

In the first step, an active site sulfhydryl performs a

nucleophilic attack on carbon 1 of glyceraldehyde-3-
phosphate, forming a covalent thioester
intermediate. The hydrogen on carbon 1 is donated
to an NAD+ cofactor in the active site, forming
NADH. An inorganic phosphate then performs a
nucleophilic attack on carbon 1, displacing the sulfur
as a sulfanion, which is quickly protonated by
another enzyme group. 1,3-bisphosphoglycerate is
now free to diffuse out of the active site.
85
 Fig. 3.34: Catalytic
mechanism of
Glyceraldehyde- 3-
phosphate
Dehydrogenase

86
 2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase

Active site details

In the animation, you can see the active site

of glyceraldehyde-3-phosphate
dehydrogenase.

87
 Fig. 3..35: Active site details of Glyceraldehyde- 3-phosphate
88 Dehydrogenase
 2. 7. Phosphoglycerate Kinase

DEPHOSPHORYLATION OF 1,3-BISPHOSPHOGLYCERATE
TO FORM 3-PHOSPHOGLYCERATE

In the seventh reaction of the glycolytic pathway,

phosphoglycerate kinase dephosphorylates 1,3-
bisphosphoglycerate to form 3-phosphoglycerate.
The free energy released in this reaction is used to
drive the formation of ATP, as 1,3-
bisphosphoglycerate donates its phosphoryl group to
ADP. Note that phosphoglycerate kinase transfers a
phosphoryl group to ADP to form ATP, whereas
hexokinase and PFK catalyze phosphoryl group
transfers from ATP.
89
 2. 7. Phosphoglycerate Kinase

Structure

Each subunit of phosphoglycerate kinase is

composed of two subdomains made of a central β
sheet surrounded by a bundle of α helices. The
active site is divided between the two domains,
shown here with the ADP bound to one domain and
1,2-bisphosphoglycerate, which is a substrate
analog, bound to the other domain.

90
91 Fig. 3.36: Structure of Phosphoglycerate Kinase
 2. 7. Phosphoglycerate Kinase

Catalytic mechanism
The mechanism is nearly the reverse of the first and
third reactions in the glycolytic pathway, that of
hexokinase and phosphofructokinase. In the first
step of the mechanism, an oxyanion of the β
phosphate of ADP performs a nucleophilic attack on
the carbon 1 phosphoester of 1,3-
bisphosphoglycerate. This leaves ATP and 3-
phosphoglycerate.

92
 Fig. 3.37: Catalytic
mechanism of
Phosphoglycerate
Kinase

93
 2. 7. Phosphoglycerate Kinase

Active site details

The illustration shows the active site of

phosphoglycerate kinase. In this case, it is
complexed with 3-phosphyglycerate and
ATP, the products of the reaction.

94
95 Fig. 3.38: Active site details of Phosphoglycerate Kinase
 2. 8. Phosphoglycerate Mutase

REARRANGEMENT OF 3-PHOSPHOGLYCERATE
TO 2-PHOSPHOGLYCERATE

In the eighth reaction of glycolysis, 3-

phosphoglycerate is converted to 2-
phosphoglycerate by the enzyme
phosphoglycerate mutase. This reaction is
an isomerization.

96
 2.8. Phosphoglycerate Mutase

Structure

Each subunit of phosphoglycerate mutase is

composed of two subdomains that are made
from a central β sheet surrounded by a
helices. The active site is located between
the two subdomains.

97
 Fig. 3.39: Structure of Phosphoglycerate Mutase
98
 2.8. Phosphoglycerate Mutase

Catalytic mechanism

The mechanism of phosphoglycerate mutase

involves creation of 2,3-bisphosphoglycerate by a
phosphorylated histidine in the enzyme. The
histidine-bound phosphate is donated to carbon-2 of
3-phosphoglycerate, then the phosphate of carbon-3
is removed by the histidine, leaving 2-
phosphoglycerate and phosphohistidine.

99
 Fig. 3.40: Catalytic
mechanism of
Phosphoglycerate
Mutase

10
 2. 8. Phosphoglycerate Mutase

Active site details

2-Phosphoglycerate is complexed with the

enzyme, and the active site histidine is
highlighted. Note how the histidine is located
right between the carbon 2 and carbon 3
positions.

10
 Fig. 3.41: Active site details of Phosphoglycerate Mutase
10
 2. 9. Enolase

DEHYDRATION OF 2-PHOSPHOGLYCERATE TO
PHOSPHOENOLPYRUVATE

In the ninth reaction of glycolysis, enolase

catalyzes a dehydration reaction of 2-
phosphoglycerate in which water is
eliminated, to yield phosphoenolpyruvate.

10
 2. 9. Enolase

Structure

Enolase is a homodimer with each subunit

being a β barrel surrounded by α helices,
similar to triose phosphate isomerase. The
interface between the two subunits is a flat
surface. The active sites are located within a
cavity close to the center of each subunit.
10
10 Fig. 3.42: Structure of Enolase
 2. 9. Enolase

Catalytic mechanism

The dehydration is initiated by a base that

abstracts a proton from carbon 2 of 2-
phosphoglycerate. This forms a carbanion
whose electron quickly initiates an enolate
formation to carbon 3. The carbon 3 hydroxyl
group leaves phosphoenolpyruvate as water
that diffuses out of the active site.
10
 Fig. 3.43:Catalytic
mechanism of
enolase

10
 2. 9. Enolase

Active site details

2-phosphoglycerate complexed with

enolase. Notice the two bound Mg2+ atoms in
each active site (in green). These divalent
cations are required for the enzyme to bind
the substrate.

10
 Fig. 3.44: Active
site details of
Enolase

10
 2. 10. Pyruvate Kinase

DEPHOSPHORYLATION OF PHOSPHOENOLPYRUVATE TO
PYRUVATE

The tenth and final reaction of glycolysis is

catalyzed by pyruvate kinase, which converts
phosphoenolpyruvate to pyruvate as it
transfers the phosphoryl group to ADP to
yield ATP.

11
 2. 10. Pyruvate Kinase

Structure

Pyruvate kinase contains a "cupped hand“

domain made from four separate segments of β
sheet surrounded by α helices. The active site
is located inside the "cup" of the hand.

11
11 Fig. 3.45: Structure of Pyruvate Kinase
 2.10. Pyruvate Kinase

Catalytic mechanism
The mechanism of pyruvate kinase
resembles that of phosphoglycerate kinase.
In the first step of the mechanism, an
oxyanion of the β phosphate of ADP
performs a nucleophilic attack on the carbon
2 phosphoester of phosphoenolpyruvate.
This leaves ATP and pyruvate ready to
diffuse out of the active site.
11
 Fig. 3.46:
Catalytic
mechanism of
Pyruvate Kinase

11
 2. 10. Pyruvate Kinase

Active site details

The active site is situated inside the "cup" of

the hand. In this structure, pyruvate and ADP
are complexed with the enzyme.

11
11 Fig. 3.47: Active site details of Pyruvate Kinase
 Conclusion

The reactions you have just seen are catalyzed by a sequence

of 10 enzymes.

Overall, the glycolytic pathway converts one molecule of

glucose to two molecules of pyruvate. Two ATP are consumed
in the hexokinase and PFK steps. Since two glyceraldehyde-3-
phosphate molecules proceed through steps 6–10, the
reactions catalyzed by phosphoglycerate kinase and pyruvate
kinase generate four ATP, for a net total of two ATP per
glucose.
Additional free energy can be extracted from the products of
glycolysis: Pyruvate can be further oxidized by the citric acid
cycle, and the NADH produced in the glyceraldehyde-3-
phosphate dehydrogenase step can be reoxidized to produce
ATP by oxidative phosphorylation.
11
 Thank you for your kindly listening

11