3.

ENZYMES
GENERAL PROPERTIES OF ENZYMES
➢ Virtually all reactions in the body are mediated by enzymes,
w/c are protein catalysts that increase the rate of reactions
without being changed in the overall process.

➢ Among the many biologic reactions that are energetically

possible, enzymes selectively channel reactants
(substrates) into useful pathways.

➢ Enzymes thus direct all metabolic events.

General properties of enzymes . . . Cont’d
➢ Enzymes, the catalysts of biological systems, are
remarkable molecular devices that determine the
patterns of chemical transformations.
➢ They also mediate the transformation of one form of
energy into another.
➢ The most striking characteristics of enzymes are their
catalytic power and specificity.
➢ Catalysis takes place at a particular site on the enzyme
called the active site.
➢ Nearly all known enzymes are proteins.
NOMENCLATURE
❑ Each enzyme is assigned two names.
I. The first is its short, recommended name. convenient for used
everyday use.
II. The second is the more complete systematic name, which is when an
enzyme must be identified without ambiguity.
a. Recommended name
Most commonly used names have the suffix “-ase” attached to the
substrate of the reaction ex. Glucosidase and urease Or to a description
of the action performed
ex. Lactate dehydrogenase adenyl cycalase
Note some enzymes retain their original trivial names, w/c give no hint of
the associated enzymic reaction. Ex. Trypsin, pepsin……
b. Systemic name

➢ are
unambiguous
and
informative but
and are
frequently too
cumbersome to
be of general
use.
➢ In this system,
enzymes are
divided into six
major classes,
each with sub
class and sub-
subclass .
➢ Its systematic name is used when ambiguity must be minimized; it is the
name of its substrate(s) followed by a word ending in -ase specifying the type
of reaction the enzyme catalyzes according to its major group classification.

➢ the systematic functional classification and nomenclature of enzymes was

adopted by the International Union of Biochemistry and Molecular Biology
(IUBMB).
Ex. the enzyme whose alternative name is lysozyme has the
systematic name peptidoglycan N-acetylmuramoylhydrolase and the
Classification Number EC 3.2.1.17. Here “EC” stands for Enzyme
Commission, the first number (3) indicates the enzyme’s major class
(hydrolases), the second number (2) denotes its subclass
(glycosylases), the third number (1) designates its sub-subclass
(enzymes hydrolyzing O- and S-glycosyl compounds), and the
fourth number (17) is the enzyme’s arbitrarily assigned serial
number in its sub-subclass.
1. Catalysis in terms of energy changes that occurs during the reaction.

Enzymes provide an alternative, energetically favorable reaction

pathways d/t from the un-catalyzed reaction. A chemical reaction

S P will take place when a certain number of S molecule at any

given instant posses enough energy to attain an activated condition
called the “Transition state” in w/c the probability of making or
braking a chemical bond to form the product is very high.

➢The transition state is the top of the energy barrier separating the
reactants and the products.
▪Activation energy is the amount of energy required to bring all the
molecules in 1g/mol of a substrate at a given temperate to the transition
state.
▪Enzyme combines transiently with the substrate to produce a transient
state having lower energy of activation than that of substrate alone.
This results in acceleration of the reaction. Once the product are
formed, the enzyme is free to combine with another molecule of the
substrate and repeat the process.
▪Activation energy is the energy required to convert all molecules in
1mole of reacting substance from the ground state to the transition
state. Enzymes are said to reduce the magnitude of this activation
energy.
during the formation of ES complex, the S attaches itself to the
specific active sites on the enzyme molecule by reversible
interactions formed by electrostatic bond, hydrogen bond,
Vanderwaals force, hydrophobic interaction.

2. how the active site chemically facilitates catalysis.

Emil Fischer’s model Lock and Key model 1890.

Lock & key model of enzyme action implies that the active site of
the enzyme is complementary in shape to that of its substrate. i.e
the shape of the enzyme & the substrate molecule should fit each
other like a lock and key.
Kinetics of enzymes
Substrate Concentration
• One simplifying approach in kinetics experiments is to measure
the initial rate (or initial velocity), designated V0, when [S] is
much greater than the concentration of enzyme, [E].

• In a typical reaction, the enzyme may be present in nanomolar

quantities, whereas [S] may be five or six orders of magnitude
higher.
• If only the beginning of the reaction is monitored (often the first
60 seconds or less), changes in [S] can be limited to a few percent,
and [S] can be regarded as constant. V0 can then be explored as a
function of [S], which is adjusted by the investigator.
Kinetics of enzymes . . . Cont’d

• At relatively low concentrations of substrate, V0 increases almost

linearly with an increase in [S]. At higher substrate
concentrations, V0 increases by smaller and smaller amounts in
response to increases in [S].

• Finally, a point is reached beyond which increases in V0 are

vanishingly small as [S] increases. This plateau-like V0 region is
close to the maximum velocity, Vmax.
Kinetics of enzymes . . . Cont’d

Effect of substrate concentration on the initial velocity of an enzyme-catalyzed

reaction.
• Leonor Michaelis and Maud Menten in 1913 postulated that
the enzyme first combines reversibly with its substrate to
form an enzyme-substrate complex in a relatively fast
reversible step:

• The ES complex then breaks down in a slower second step to

yield the free enzyme and the reaction product P:
Steady-state Kinetics
➢ When the enzyme is first mixed with a large excess of substrate,
there is an initial period, the pre–steady state, during which the
concentration of ES builds up.
➢ This period is usually too short to be easily observed, lasting just
microseconds.
➢ The reaction quickly achieves a steady state in which [ES] (and
the concentrations of any other intermediates) remains
approximately constant over time.
➢ The concept of a steady state was introduced by G. E. Briggs and
Haldane in 1925.
➢ The measured V0 generally reflects the steady state, even though
V0 is limited to the early part of the reaction, and analysis of these
initial rates is referred to as steady-state kinetics.
Enzyme Kinetics . . .
Many Enzymes Catalyze Reactions with Two or More Substrates
➢ In most enzymatic reactions, however, two (and sometimes more)
different substrate molecules bind to the enzyme and participate in
the reaction.
➢ For example, in the reaction catalyzed by hexokinase, ATP and
glucose are the substrate molecules, and ADP and glucose 6-
phosphate are the products:
ATP + glucose ADP + glucose 6-phosphate
➢ The rates of such bi-substrate reactions can also be analyzed by
the Michaelis-Menten approach. Hexokinase has a characteristic
Km for each of its substrates.
➢ Enzymatic reactions with two substrates usually involve transfer
of an atom or a functional group from one substrate to the other.
As another example, the enzyme with the recommended name

alcohol dehydrogenase has the systematic name

alcohol:NAD+ oxidoreductase and the classification number EC 1.1.1.1.

Nature of Enzymes

Most enzymes are protein in nature. Depending on the presence and

absence of a non-protein component with the enzyme, enzymes can exist
as, simple enzyme or holo enzyme

1.Simple enzyme; is made up of only protein molecules not bound to any

non-proteins.

Ex. Pancreatic Ribonuclease.

2. Holo enzyme; is made up of protein groups and non-protein
components.
▪ The protein component is called apoenzyme
▪ The non-protein component is called a cofactor
If this Cofactor is an organic cmpd it is called a coenzyme and if it
is an inorganic groups it is called activators ( fe2+, Mn2+,
Zn2+……ions).
If the cofactor is bound so tightly to the apo-enzyme and is difficult
to remove without damaging the enzyme it is sometimes called
Prosthetic groups
 Simple enzymes Apo Enzyme
Enzymes Holo enzymes Cofactors
COENZYMES; are derivatives of vitamins without which the
enzyme cannot exhibit any reaction. One molecule of coenzyme is
able to convert a large number of substrate molecules with the help
of enzyme.

✓ coenzyme accepts a particular group removed from the substrate

or donate a particular group to the substrate
✓Coenzymes are called co-substrate b/c the changes that take place
in substrates are complimentary to the changes in coenzymes.
✓ The coenzyme may participate in forming an intermediate enzyme-
substrate complex. Ex. NAD, FAD, CoA (CoA)
Metal ions in Enzymes
Many enzymes require metal ions like Ca2+ Cu2+,, Zn2+, , Mn2+ , &
Co2+ for their activity.

Metal-activated enzymes form only loose and easily dissociable

complexes with the metal and can easily release the metal without
denaturation. Metalloenzymes hold the metal tightly on the molecule
and don’t release it even during extensive purification.
Metal ions promote enzyme action by:

a. Maintaining or producing the active structural conformation of

the enzyme. Ex. Glutamine synthase

b. Promoting the formation of the enzyme-substrate complex

Ex. Enolase and Carboxypeptidase

c. Acting as electron donors or acceptors Ex. Fe-S proteins &

Cytochromes

d. Causing distortions in the substrate or the enzyme Ex.

Phosphotransferases
Properties of Enzymes
a. Active sites
Enzyme molecules contain a special pocket or cleft called the
active site. It is formed by folding of the protein, contains a.a side
chains that participate in substrate binding and catalysis. The
substrate binds the enzyme, forming an enzyme-substrate complex
(ES). Binding is thought to cause a conformational change in the
enzyme (induced fit model) that allows catalysis. ES is converted
to an enzyme-product (EP) complex that subsequently dissociate
to enzyme and product.
b. Catalytic efficiency/ enzyme turnover number

Enzyme-catalyzed reactions are highly efficient, proceeding from

103-108 times faster than un-catalyzed reactions. Typically each
enzyme molecule is capable of transforming 100 to 1000 substrate
molecule into product each second The number of molecules of
substrate converted to product per enzyme molecule per second is
called turn over number, and typically is 102- 104s-1 ( Carbonic
anhydrase is the fastest enzyme).
c. Specificity
Enzymes are highly specific, interacting with one or a few substrates
and catalyzing only one type of chemical reaction. The set of enzymes
made in a cell determines which reactions occur in that cell. Specificity
of enzymes are either:
a. Absolute; this means one enzyme catalyzes or acts on only one
substrate. Ex. Urease catalyzes hydrolysis of urea but not thiourea.
b. Stereo; some enzymes are specific to only one isomer even if the
cmpd is one type of molecule. Ex. Glucose oxidase catalyzes the
oxidation of β-D-glucose but not σ-D- glucose.
Bond specificity; ex. Esterases---acts on ester bonds
Peptidases---acts on peptide bonds
Glycosidases---acts on glycosidic bonds.
D. Regulation
Enzyme activity can be regulated- that is, enzyme can be, activated or
inhibited so that the rate of product formation responds to the needs
of the cell.
E. Zymogens (-inactive form of enzymes)
Some enzymes are produced in nature in an inactive form which can
be activated when they are required such enzymes are called
Zymogens (proenzymes).
Ex. Pepsinogen- when required converted to Pepsin.
Pepsinogen + H+ Pepsin
Trypsinogen- when required converted to trypsin.
Trypsinogen Enteropeptidase Trypsin
Zymogen forms of enzymes a protective mechanism to prevent auto
digestion of tissue producing the digestive enzymes and to prevent
intravascular coagulation of blood.
F. Iso-enzymes (Isozymes)
These are enzymes having similar catalytic activity, act on the same
substrate and produces the same product but originated at different
site and exhibiting d/t physical & chemical x-stics such as
electrophoretic mobility, a.a composition & immunological
behavior.
Ex. LDH (Lactate dehydrogenase) exists in five d/t forms each
having four polypeptide chains
How enzymes work
The mechanism of enzyme action can be viewed from two d/t
perspectives.
Factors affecting rate of enzyme catalyzed reaction
Physical and chemical factors are affecting the enzyme activity.
These include:

1. Temperature

Staring from low temp. as the temp. increases to certain degree

the activity of the enzyme increases b/c the temp. increases the
total energy of the chemical system.

There is an optimal temp. at w/c the reaction is most rapid but

above this the reaction rate decreases sharply, maily due to
denaturation of the enzyme by heat.
2. Effect of PH
The concentration of H+ affects reaction velocity in several ways.
First the catalytic process usually requires that the enzyme and
substrate have several specific chemical groups in an ionized or
unionized state in order to interact

Ex. Catalytic activity may require that an amino-group of the

enzyme be in the protonated form (NH3+) at alkaline PH this
group is deprotonated and the rate of reaction therefore declines.

3. Concentration of substrate

At fixed enzyme concentration PH & Temp. the activity of

enzymes is influenced by increase in substrate concentration.
This condition shows that as concentration of substrate is increased,
the substrate molecule combines with all available enzyme
molecules at their active site till not more active sites are available
at this state the enzyme is obtained its maximum rate (Vmax)
Vmax………………………………………………………….

(S)
The x-stics shape of the substrate saturation curve for an enzyme
can be expressed mathematically by the Michaelis Menten
equation.
V= Vmax [S] V= velocity at a given concentration
Km + [S] Vmax= maximum velocity possible with excess of substrate
[S] = concentration of the substrate at velocity
km= Michaells-constant of the enzyme for particular substrate

Relation ship between [s] and km

km shows the r/s b/n the substrate concentration and the velocity of
the enzyme catalyzed reaction. Take the point in w/c 50% of the
active site of the enzyme will be saturated by substrate, assume that at
½ Vmax-50% of the active site of an enzyme become saturated.
Therefore:
V0 = ½ Vmax, at 50% saturation
½ Vmax = Vmax[s]/km + [S]
2[S] = km + [S]
km = [S]

Vmax……………………………………..

V ………………………………

½ Vmax

km [S]
 INHIBITION OF ENZYME ACTIVITY
Any substance that can decrease the velocity of the enzyme-catalyzed
reaction is called an inhibitor and the process is known as inhibition.
There are two major types of enzyme inhibition:
1. Irreversible inhibition
the type of inhibition that can not be reversed by increasing substrate
concentration or removing the remaining free inhibitors.
Ex. Pb2+ forms a covalent bond with the sulfhydryl side chain of cysteine
in protein.
Ferrochelatase an enzyne involved in heme synthesis is irreversably
inhibited by Pb2+
Diisopropyl & luorophosphate (DFP) inhibits trypsin, chymotrypsin,
elastase & phosphoglucomutase
Irreversible inhibitors also referred to as “suicide inhibitors”
2. Reversible inhibitors
Bind to enzymes through non-covalent bonds and thus dilution of the
enzyme-inhibitor complex results in dissociation of the reversibly
bound inhibitor and recovery of enzyme activity. The two most
commonly encountered types of reversible inhibition are competitive
and non competitive.
A. Competitive inhibition
this type of inhibition occurs when the inhibitor binds reversibly to
the same site that the substrate would normally occupy and,
therefore, competes with the substrate for the site.
The inhibitor and the substrate compete for the same active site on the
enzyme as a result of similarity in structure. The enzyme substrate
complex will be broken dawn to products ( E + S ES E + P)
where as enzyme inhibitor complex (EI) will not be broken down
to products. Ex. Malonate that compete with succinate and
inhibits the action of succinate dehydrogenase to produce
fumarate in the krebs cycle.
The effect of competitive inhibition is reversed by increasing [S]. At
a sufficiently high substrate concentration, the reaction velocity
reaches the vmax observed in the absence of inhibitor.
B. Non-competitive inhibition
This type of inhibition is recognized by its characteristics effect on
vmax non-competitive inhibition occurs when the inhibitor and
substrate bind at d/t sites on the enzyme. The non-competitive
inhibitors can bind either free enzyme or the enzyme-substrate
complex, thereby preventing the reaction from occurring.
Non-competitive inhibition can not be overcome by increasing
concentration of substrate. Therefore, noncompetitive inhibitors
decrease the apparent vmax of the reaction.
 REGULATION OF ENZYME ACTIVITY
The regulation of the reaction velocity of enzymes is essential if an
organism is to coordinate its numerous metabolic processes. The
rates of most enzymes are responsive to changes in substrate
concentration, b/c the intercellular level of many substrate is in the
range of the km. thus, an increase in substrate concentration prompts
an increase in reaction rate, w/c tends to return the concentration of
substrate toward normal.
In addition, some enzymes with specialized regulatory functions responds to
allosteric effectors and/or covalent modification or they show altered rates of
enzyme synthesis when physiologic conditions are changed.
A. Regulation of allosteric enzymes
Allosteric enzymes are regulated by molecules called effectors that
bind non-covalently at a site other than the active site. These
enzymes are almost always composed of multiple sub units, and
the regulatory (Allosteric) site that binds the effector is distinct
from the substrate-binding site and may be located on a sub unit
that is not itself catalytic. Effectors that inhibits enzyme activity
are termed negative effectors, whereas those that increase enzyme
activity are called positive effectors.
B. Regulation of enzymes by covalent modifications
Many enzymes are regulated by covalent modifications, most often by
the addition or removal of phosphate groups from specific serine,
therionine, or tyrosine residues of the enzyme. Protein phosphorylation
is recognized as one of the primary ways in w/c cellular process are
regulated.

C. Induced and repression of enzyme synthesis

Cells can also regulate the amount of enzyme present by altering the rate of
enzyme degradation or, more typically, the rate of enzyme synthesis. The
increase (induction) or decrease(repression) of enzyme synthesis leads to
an alteration in the total population of active sites.
Important characteristics of enzymes:

Specificity
May require: activation
Cofactors or Coenzymes
ENZYMES IN CLINICAL DIAGNOSIS
Plasma enzymes can be classified into two major groups
1. Those, relatively, small group of enzymes secreted into
the plasma by certain organs (i.e. Enzymes those have
function in plasma) For example: - the liver
secretes zymogens of the enzymes involved in blood
coagulation.
2. Those large enzyme species released from cells during
normal cell turnover.
These enzymes are normally intracellular and have no
physiologic function in the plasma.
Measurement of enzymes concentration of mostly the latter type in plasma
gives valuable information about disease involving tissues of their origin

1. Lipase: It is an enzyme catalyzing the hydrolysis of fats. It is

secreted by pancreas and Liver.
2. α- Amylase: break down of dietary starch and glycogen to
maltose. It is present in pancreatic juice and saliva as well as in
liver fallopian tubes and muscles.
3. Trypsin: Trypsin is secreted by pancreas. Elevated levels of
trypsin in plasma occur during acute pancreatic disease.
4. Alkaline phosphates (ALP): hydrolyze phosphate esters
at an alkaline pH. found in bone, liver, kidney, intestinal wall,
lactating mammary gland and placenta.
5. Acid Phosphatase (ACP): hydrolysis of various
phosphate esters at acidic pH is found in the prostate, liver, red
cells, platelets and bone
Measurement of enzymes concentration of mostly the latter type in plasma
gives valuable information about disease involving tissues of their origin

6. Transaminases:
1. Asapartate transaminase AST, ( Glutamate oxaloacetate
transaminase, GOT )
❑ catalyzes the transfer of the amino group of aspartic acid to α-
ketoglutarate forming glutamate and oxaloacetate
2. Alanine transaminase, ALT (Glutamate pyruvate
transaminase, GPT )
❑ Transfer the amino group of alanine to α- ketoglutarate, forming
glutamate and pyruvate.
7. Lactate Dehydrogenase (LDH):
It catalyzes the reversible interconversion of lactate and pyruvate.
8. Creatine kinase (CK) or ceratin phosphokinase (CPK)
CK (CPK) is found in heart muscle brain and skeletal muscle.
4. CENTRAL METABOLISM

➢Metabolism is thus the sum of chemical reactions that

takes place within each cell of a living organism and that
provides energy for vital processes and synthesizing of
new organic materials.
Broadly, these reactions can be divided into
A. catabolic reactions that convert nutrients to energy
and
B. anabolic reactions that lead to the synthesis of larger
biomolecules.
 CELLULAR METABOLISM . . . CONT’D

Metabolic Pathways
➢a series of chemical reactions in which the product of one
reaction is the substrate for the next reaction.
Catabolic pathways: release energy by breaking down larger
molecules into smaller molecules.
Anabolic pathways: use the energy released by catabolic
pathways to build larger molecules from smaller molecules,
ensuring the continual flow of energy within an organism.
Metabolites: are the reactants and products of chemical reactions.
A Biochemical Pathway
 CENTRAL METABOLISM . . . CONT’D

Metabolites
➢The major classes of metabolites include proteins, carbohydrates,
nucleotides, lipids, coenzymes, and cofactors.
➢encompass an enormous diversity of molecular structures,
physicochemical properties, functions, and abundances.
Polar (Hydrophilic Metabolites):
❑are soluble in aqueous solutions.
❑include most of the reactants and products involved in cellular
respiration and in the synthesis of large biopolymers.
▪ include most sugars, purines and pyrimidines, nucleotides and nucleosides,
acyl carnitines, organic acids, hydrophilic acids, amino acids, and
phosphorylated compounds.
 CENTRAL METABOLISM . . . CONT’D

Metabolites
Non-polar (Hydrophobic Metabolites):
❑are commonly lipids.
❑function in energy storage, membrane structure, and signal
transduction.
CELLULAR RESPIRATION
▪ Cellular respiration is a process of breaking down organic molecules
(catabolism) to harvest electrons from carbon compounds, such as glucose, and
use that energy to make Adenosine Tri Phosphate (ATP).
▪ ATP is used to provide immediate energy for cells to do work
Divided into 3 phases
 Phase I: Breakdown of large complex biomolecules like polysaccharides,
proteins and lipids into their respective building blocks (hydrolysis).
 Phase II: These building blocks are usually oxidized to a common intermediate,
acetyl - CoA.
 Phase III:This consists of the citric acid cycle (i.e. oxidation of acetyl - CoA to
CO2, formation of NADH and FADH2) followed by electron transport and
oxidative phosphorylation.
 CELLULAR RESPIRATION
ATP STRUCTURE

ATP is
▪Energy used by all Cells
▪Adenosine
Triphosphate
▪Organic molecule
containing high-energy
Phosphate bonds
GLYCOLYSIS: ANAEROBIC RESPIRATION

➢During glycolysis, glucose molecules (six-carbon molecules) are split

into two pyruvates (three-carbon molecules) during a sequence of
enzyme-controlled reactions.

➢This is the same reaction as occurs in aerobic respiration. Without

oxygen, pyruvate is converted to lactic acid in animals or ethanol in
plants and yeast.
➢Glycolysis is the sequence of ten enzymatic reactions that oxidize the
six-carbon sugar glucose into two three-carbon compounds (Pyruvate)
with the production of a small amount of adenosine tri phosphate (ATP).
➢In the process, glycolysis produced four ATP for a net gain of two ATP
and two molecules of NADH.
SOME FATES OF GLUCOSE
THE STAGES OF GLUCOSE METABOLISM
Glycolysis
Phosphorylation
1. During phosphorylation glucose is converted into
glucose 6-phosphate using enzyme hexokinase
phosphorylate (adds a phosphate group ) to glucose.
2. The enzyme phosphoglucoisomerase converts glucose
6-phosphate into its isomer fructose 6-phosphate
3. The enzyme phosphofructokinase (PFK) uses another
ATP molecule to transfer a phosphate group to fructose
6-phosphate to form fructose 1, 6-bisphosphate.
Glycolysis . . . Cont’d

4. The enzyme aldolase splits fructose 1, 6-bisphosphate into

two triose sugars that are isomers of each other.
These two sugars are
- dihydroxyacetone phosphate and
- glyceraldehyde-3- phosphate.
❖Only, Glycelaldehyde-3-phosphate proceeds immediately
through glycolysis.
❖Whereas, Dihydroxyacetone phosphate is converted into
glyceraldehyde-3- phosphate by trios phosphate isomerase
Glycolysis . . . Cont’d

5. The enzyme triose phosphate isomerase rapidly inter-converts

the molecules dihydroxyacetone phosphate into
glyceraldehyde-3-phosphate. Glyceraldehyde phosphate is
removed as soon as it is formed to be used in the next step of
glycolysis.
6. The enzyme triose phosphate dehydrogenase serves two
functions in this step.
❖First the enzyme transfers hydrogen (H-) from glyceraldehyde
phosphate to the oxidizing agent Nicotinamide Adenine
Dinucleotide (NAD+) to form NADH. The other one is triose
phosphate dehydrogenase adds a phosphate (P) from the cytosol to
the oxidized glyceraldehyde phosphate to form 1, 3-
diphoshoglyceric acid.
Glycolysis . . . Cont’d

7. The enzyme phosphoglycerokinase or

phosphoglycerate Kinase transfers a Phosphate (P)
from 1, 3-diphoshoglyceric acid to a molecule of
ADP to form ATP. This happens for each molecule of
1, 3-diphoshoglyceric acid.
▪ The process yields two 3-phosphoglyceric acid molecules and
two ATP molecules. The first time in history of glycolysis ATP
is harvested.

8. The enzyme phosphoglyceromutase relocates the P

from 3-phosphoglyceric acid from the third carbon
relatively low free energy of hydrolysis to the second
carbon to form 2-phosphoglyceric acid.
Glycolysis . . . Cont’d

9. The enzyme Enolase removes a molecule of water

from 2-phosphoglyceric acid to form high energy
phosphoenolpyruvic acid (PEP).
10.The enzyme pyruvate kinase transfers a P from PEP
to ADP to form pyruvic acid and ATP. This happens
for each molecule of PEP. This reaction yields two
molecules of pyruvic acid and two ATP molecules
THE LINK REACTION

➢This links glycolysis to the Krebs Cycle (sometimes called the

citric acid cycle).

➢Pyruvate molecules are decarboxylated (they lose a molecule

of carbon dioxide) in the mitochondria. Pyruvate molecules are
oxidized and converted to acetylcoenzyme A, usually
abbreviated to acetyl CoA.

➢The oxidised form of nicotinamide adenine dinucleotide,

NAD+, is reduced to its reduced from NADH.
LINK REACTION
 THE KREBS CYCLE

▪ It occurs in the matrix (fluid portion) of the mitochondrion. Acetyl CoA (2 carbon
cpds) produced by the link reaction joins oxaloacetate (4 carbon cpds)
▪ Turns twice per glucose molecule
Functions of the Kreb Cycle:
• H+ and e- are transferred to NAD+ and FAD to become NADH and FADH2,
respectively, two molecules of ATP are produced by substrate-level
phosphorylation and Most of the molecules are recycled to conserve oxaloacetate
for continuous processing of acetyl-CoA.
▪ Substrate-level phosphorylation is a type of chemical reaction that results in the
formation of adenosine triphosphate (ATP) by the direct transfer of a phosphate
group to adenosine diphosphate (ADP) from a reactive intermediate. In cells, it
occurs in the cytoplasm (in glycolysis) and the mitochondrial matrix (in the citric
acid cycle) under both aerobic and anaerobic conditions.
STEPS IN KREBS CYCLE

1. The acetic acid subunit of acetyl CoA (two carbons) is combined with
oxaloacetate (four carbons) to form a molecule of citrate (six
carbons). The acetyl coenzyme A acts only as a transporter of acetic
acid from one enzyme to another.
2. A hydroxyl group and a hydrogen molecule are removed from the
citrate structure in the form of water. isocitrate is formed. It is
catalyzed by an enzyme, Aconitase. the hydroxyl group and hydrogen
molecule are reversed with respect to the original structure of the
citrate molecule
3. The isocitrate molecule is oxidized by a NAD+ molecule. The NAD+
molecule is reduced by the hydrogen atom and the hydroxyl group. The
NAD+ binds with a hydrogen atom and carries off the other hydrogen
atom leaving a carbonyl group. This structure is very unstable, so a
molecule of CO2 is released creating alpha-ketoglutarate (5 carbon
cpds). It is catalyzed by an enzyme, Isocitrate dehydrogenase.
 STEPS IN KREBS CYCLE . . . CONT’D

4.coenzyme A, returns to oxidize the alpha-ketoglutarate molecule. A

molecule of NAD+ is reduced again to form NADH and leaves with
another hydrogen. This instability causes a carbonyl group to be released
as carbon dioxide and a thioester bond is formed in its place between the
former alpha-ketoglutarate and coenzyme A to create a molecule of
succinyl-coenzyme A complex (Four carbon cpds). It is catalyzed by an
enzyme 2-Ketoglutarate dehydrogenase complex.
5. A water molecule sheds its hydrogen atoms to coenzyme A. Then, a free-
floating phosphate group displaces coenzyme A and forms a bond with the
succinyl complex. The phosphate is then transferred to a molecule of GDP
to produce an energy rich molecule of GTP. It leaves behind a molecule of
succinate. It is catalyzed by an enzyme Succinyl-CoA synthetase.
 STEPS IN KREBS CYCLE

6. In this step, succinate is oxidized by a molecule of FAD (Flavin

adenine dinucleotide). The FAD removes two hydrogen atoms from
the succinate and forces a double bond to form between the two carbon
atoms, thus creating fumarate. It is catalyzed by an enzyme succinate
dehydrogenase.

7. An enzyme adds water to the fumarate molecule to form malate. The

malate is created by adding one hydrogen atom to a carbon atom and
then adding a hydroxyl group to a carbon next to a terminal carbonyl
group. It is catalyzed by an enzyme Fumarate hydratase.
STEPS IN KREBS CYCLE

8. In this final step, the malate molecule is oxidized by a

NAD+ molecule. The carbon that carried the hydroxyl
group is now converted into a carbonyl group. The end
product is oxaloacetate which can then combines with
acetyl-coenzyme A and begin the Krebs cycle all over
again. It is catalyzed by an enzyme Malate
dehydrogenase.
THE KREBS CYCLE
Functions of Kreb’s cycle
Kreb’s cycle has catabolic and anabolic functions:
✓ Energy generation- Reducing equivalents, NADH, FADH2 & ATP
✓ Provide CO2 used for – gluconeogenesis
- Fatty acid synthesis
- Urea synthesis
- Nucleotide synthesis
✓ Provide precursors for:
- gluconeogenesis ( all intermediates)
- amino acid synthesis ( non-essential a.a)
- heme synthesis ( succinyl CoA)
- fatty acid synthesis (Citrate)
✓ Regulate other pathways – Citrate ( inhibit phosphofructokinase)
Electron Transport System (ETS)

➢ETS takes place in the inner membrane of mitochondria.

➢The NADH and FADH2 formed in glycolysis, fatty acid
oxidation and the citric acid cycle are energy-rich molecules
because each contains a pair of electrons having a high transfer
potential.
➢When these electrons are used to reduce molecular oxygen into
water, a large amount of free energy is liberated, which can be
used to generate ATP the process is called oxidative
phosphorylation.
Electron Transport System (ETS)

➢Proteins imbedded in the inner membrane are use for

transfer of these electrons from higher energy state to
lower energy state and finally accepted by molecular
oxygen
➢can be used to phosphorylate ADP
➢Two energy carriers are known to donate energy to the
ETS, namely nicotine adenine dinucleotide (NAD) and
flavin adenine dinucleotide (FAD).
➢cytochrome oxidase, uses the energy it receives along
with an electron pair to reduce oxygen to water
OXIDATIVE PHOSPHORYLATION

➢NADH 'carries' hydrogen ions and high-energy electrons. In

oxidative phosphorylation the hydrogen ions combine with
oxygen to form water and the electrons pass along an
electron transfer chain (also called the respiratory chain)
using their energy to form ATP molecules. One molecule of
NADH forms three ATP molecules.
➢ATP production is greatly increased by oxygen. By
combining with hydrogen ions (and accepting electrons) to
form water it allows more hydrogen ions to be released from
the electron carrier system.
WHAT CARRIES THE ELECTRONS?

 NAD+ (nicotinadenine
dinucleotide) acts as
the energy carrier
 NAD+ is a coenzyme
 It’s Reduced to NADH
when it picks up two
electrons and one
hydrogen ion
 FAD+ (Flavin adenine
dinucleotide)
 Reduced to FADH2
 DIAGRAM OF THE PROCESS
Occurs
across
Cristae

Occurs in
Cytoplasm

Occurs in
Matrix
SUMMARY OF PRODUCTS FROM COMPLETE AEROBIC CELLULAR
RESPIRATION OF ONE GLUCOSE

Stage CO2 NADH FADH2 ATP

Glycolysis No Two No 4 ATP, net 2
Two Link Two mole Two No No
Two TCA Cycle Four mole Six Two Two
ETC Glycerol phosphate shuttle: Total ATP:
No
Glycolysis, 2 NADH x 2 = 4 ATP 32 ATP
2 Link, 2NADH X 3 = 6 ATP
6 TCA, 6 NADH x 3 = 18 ATP
2 TCA, 2 FADH2 x 2 = 4 ATP
Malate aspartate shuttle: Total ATP
Glycolysis, 2 NADH x 3 = 6 ATP 34 ATP
2 Link, 2NADH X 3 = 6 ATP
6 TCA, 6 NADH x 3 = 18 ATP
2 TCA, 2 FADH2 x 2 = 4 ATP
Total ATP Produced will be 38 (if NADH of glycolysis is
transported by malate aspartate shuttle) or 36 Glycerol
phosphate shuttle.
Bioenergetics
✓ describes the transfer & utilization of energy in biologic systems.

(Particularly the concept of free energy)

✓ changes in free energy provide a measure of the energetic feasibility

of a chemical reaction &, can therefore, allow prediction of whether the
reaction or a process can take place.

✓ Bioenergetics concerns only the initial & final energy states of

reaction components, not the mechanisms or how much time is needed
for the chemical change to take place (the rate)

✓ in short bioenergetics predicts if a process is possible whereas

kinetics measures how fast the reaction occurs.
 FREE ENERGY (G)
The direction & extent to w/c a chemical reaction proceeds is
determined by the degree to w/c two factors change during the reaction.
These are:
Enthalpy (ΔH, a measure of the change in heat content of the reactants
and products)
Entropy (ΔS, a measure of a change in randomness or disorder of
reactants & products).
ΔG = ΔH - T ΔS (ΔG = energy available to do work
approaches zero as reaction proceeds to equilibrium
predicts weather the reaction is favorable
ΔH = Heat released or absorbed during the reaction.
does not predict weather the reaction is favorable.
ΔS = measure a randomness
does not predict weather the reaction is favorable.
T = is the absolute temperature
 A B B A
Transition state Transition state

------------------------- A
initial state ---------------------------------
(G) ΔG is negative final state

(G) ΔG is positive
-----------------------------B ----------------------------------
final state initial state

negative ΔG = there is a net loss of energy, & the reaction goes

spontaneously A B the reaction is said to be exergonic
Positive ΔG = there is a net gain of energy, & the reaction does not
go spontaneously from B A energy must be added to the system to
make the reaction go, the reaction is said to be endergonic.
 a. Phosphoryl group transfer and ATP

✓ At pH 7, ATP – carries 4 negative charges and repel each other

✓ repulsion is relieved upon hydrolysis of high- energy bonds

✓ATP and other high energy compounds contain phosphoanhydride

bonds w/c release much free energy upon hydrolysis.
 b. Energy of hydrolysis of phosphate bonds
▪ hydrolysis of high-energy phosphate bond of ATP releases free energy
of about -7.3kcal/mol
▪ The energy released may result in:
➢ transfer of phosphate group with partial conservation of energy by
newly formed bond formation of new bond
➢ transport molecules across membranes
➢ some portion lost as heat ( may contribute to body temp. maintenance
in homoeothermic organisms).
➢ signal amplification
➢ Change in conformation of molecules
ATP + H2O ADP + pi + energy
ATP + Substrate ADP + substrate-phosphate + heat
 c. Cellular formation and utilization of ATP
• Within cells ATP is continuously formed and utilized.
• Serve as the principal immediate donor of free energy in biological system
Oxidation of fuel molecules
▪ Catabolism of fuel molecules occurs stepwise each step releasing partial
energy content of molecules.
▪ The amount of total energy release depends upon the cellular conditions
I. presence or absence of oxygen (aerobic or anaerobic)
II. presence or absence of specific organelles with oxidative functions
(mitochondrial)
▪ Catabolic reactions in addition, provide building blocks for biosynthetic
reactions.
 An Overview of Catabolism of Fuel Molecules
a. Carbohydrate-digestion or Mobilization of Glycogen
Monosaccharide's

Cellular Catabolism Glycolysis

Kreb’s cycle Energy, CO2
Electron transport chain & H2O
Exception- RBC undergo only Glycolysis
Glycolysis - Partial catabolism
- Small amount of energy conserved (ATP, NADH)
- Prepares carbohydrates for the next catabolic processes
- sometimes the only life sustaining energy generating process
- Exercising muscle (Oxygen limitation)
b. Fats-digestion or mobilization of stored fat
Fatty acids + Glycerol
Transport by albumin
Oxidation by major pathway β-oxidation
- NADH
-FADH2
- Acetyl CoA- common intermediate
NB. No direct high-energy phosphate molecule is formed
c. Protein digestion or mobilization of tissue protein
Glycolytic intermediate Further catabolism
Acetyl CoA
Amino acids Kreb’s cycle intermediate Energy &
building blocks
During cellular oxidation of fuel molecules very little energy is directly conserved in the
form of high energy phosphate bond that can be directly utilized for cellular energy
requiring processes. Most of it is captured in the form of reducing equivalents such as
NADH ( reduced nicotinamide adenosine dinucleotide) & FADH2 (reduced flavin
adenosine dinucleotide)
d. Oxidation- Reduction Reaction
The utilization of chemical energy in living system involves oxidation-reduction
reactions. Ex. The energy of chemical bonds of carbohydrates, lipids and proteins is
released and captured in utilizations form by processes involving oxidations-reductions.
I. Oxidation- removal of electrons from substance usually accompanied
by a decrease in energy content of oxidized substance
II. Reduction- addition of electrons to substance usually accompanied by an increase
in energy content of reduced substances.
Note. Oxidation reduction reactions are coupled processes
Example: H2 2e- + 2H+ - first half reaction
- Oxidation of H2
- release electrons and protons
- requires second half reaction
½ O2 + 2e- + 2H+ H2O – second half reaction
- reduction of O2
- Oxidation of H2 is coupled to reduction of O2
➢ Most molecules serve as both electron donors and electron acceptors
of d/t times depending upon what other substances they react with.
➢ In biological systems the primary electron donors are fuel molecules
such as carbohydrates, fats and proteins.
➢ The oxidation of these substances transfers electrons to intermediate
electron carriers such as NAD+, NADP+, & FAD to reduce them to their
reduced state NADH, & FADH2.
Mitochondria- is an organelle where major amounts of energy produced.
structurally it is bounded by two separate membranes.
➢ Outer membrane - smooth and unfolded
- Freely permeable to most ions and polar molecules
(Contain porous channels)
➢ Inner membrane - Folded into Cristae-increased surface area
- highly impermeable to most ions & polar molecules
➢ Contains transporters w/c access polar & ionic molecules in &out
Cristae are characteristic of muscle and other metabolically active cell types,
about 75% protein rich membrane.
➢ Inter membrane space – space b/n outer and inner membranes
➢ Matrix- the internal compartment containing soluble enzymes and
mitochondrial genetic material (mitochondrial ribosome, DNA Granules).
Phosphocreatine system
✓ Creatine phosphate or phosphocreatine (PCr). is one of the immediate
source of energy for high intensity exercise it is also a high-energy
phosphate in which a single phosphate molecule is attached to a molecule
of creatine.
✓ Hydrolysis of PCr is brought about by the enzyme creatine kinase as follows:
ADP + PCr ATP + Creatinine = PCR Creatine + pi + energy (43kj)
Ck Ck
1 mole PCr 1 mole ATP
✓ Releases energy more rapidly than other system
✓ Used in activities that require only a few seconds (not more than 10s)
✓ No lactic acid is produced
kas@2009 94
 5. Carbohydrates
Carbohydrates or saccharides (Greek: sakcharon, sugar) are
essential components of all living organisms and are, in fact, the
most abundant class of biological molecules. The name
carbohydrate, which literally means “carbon hydrate,” stems from

their chemical composition, which is roughly

(C.H2O)n, where n is ≥3. The basic units of carbohydrates
are known as monosaccharides. Many of these compounds are
synthesized from simpler substances in a process named
gluconeogenesis.
CARBOHYDRATE

Linking Sugars Together Sugars can be joined to one

another by covalent glycosidic bonds to form larger molecules.
• Glycosidic bonds form by reaction between carbon atom
C1 of one sugar and the hydroxyl group of another sugar,
generating a —C—O—C— linkage between the two
sugars.
• sugars can be joined by quite a variety of different
glycosidic bonds.
CARBOHYDRATES

Figure 2.2 Aldotetroses. Because they have two asymmetric

carbon atoms, aldotetroses can exist in four configurations.
Reading assignment
Stereoisomerism
The metabolic breakdown of Carbohydrates:
✓provides much of the energy used to power biological processes.
✓principal components of nucleic acids, as well as important elements of
complex lipids
✓Acting as a storage form of energy in the body
✓ serving as cell membrane components that mediate some forms of
intercellular communication.
✓Serve as a structural component of many organisms, exoskeleton of many
insects, and the fibrous cellulose of plants
Classification of Carbohydrates
There are three major classes of carbohydrates
Monosaccharides (one), Oligosaccharide (few), Polysaccharides (many).
The structures of sugars. (a) Straight-chain formula of fructose, a ketohexose
[keto, indicating the carbonyl (yellow), is located internally, and hexose
because it consists of six carbons]. (b) Straight chain formula of glucose, an
aldohexose (aldo because the carbonyl is located at the end of the molecule).
(c) Self-reaction in which glucose is converted from an open chain to a closed
ring (a pyranose ring).
1. Monosaccharides ( Simple sugar)
Monosaccharides can be classified according to the number of
carbon atoms they contain as:
Generic name Example
➢ 3 carbons Trioses Glyceraldehde
➢ 4 carbons Tetroses Erythrose
➢ 5 carbons pentoses Ribose
➢ 6 carbons Hexoses Glucose
➢ 7 carbons Heptoses Sedoheptulose
➢ 9 carbons nonoses Neuraminic acid

They can also be classified by the type of carbonyl group they contain:
❖ Aldoses: Carbohydrates with an aldehyde as their carbonyl group
Ex. Glyceraldehyde

❖ Ketoses: Carbohydrates with a ketone as their carbonyl group

Ex. Dihydroxyacetone
CARBOHYDRATES

Monosaccharides
➢Monosaccharides containing the aldehyde group are
classified as aldoses, and those with a ketone group are
classified as ketoses.
➢ in water, pentoses and hexoses exist mainly in the cyclic
form, and it is in this form that they combine to form larger
saccharide molecules.
a. Glucose_important carbohydrate fuel in human cells
➢Two glucose molecules react to form the disaccharide
maltose. Starch and cellulose are polysaccharides made up
of glucose units.
CARBOHYDRATES
b. galactose
➢Galactose molecules look very similar to glucose molecules.
➢They can also exist in α and β forms. Galactose reacts with
glucose to make the disaccharide lactose.
➢However, glucose and galactose cannot be easily converted into
one another.
c. fructose
➢glucose and galactose are aldoses (reducing sugars), fructose
is a ketose (a non-reducing sugar).
➢It also has a five-atom ring rather than a six-atom ring.
Fructose reacts with glucose to make the disaccharide sucrose.
Properties of Monosaccharide
Physically monosaccharide are colorless, crystalline compounds, readily
soluble in water.
Asymmetric Center and Stereoisomerism
a. Isomers and Epimers
Cmpds that have the same chemical formula but have d/t structures
are called isomers. Ex. Fructose, glucose, mannose and galactose
are all isomers of each other, having the same chemical formula,
C6H12O6. Carbohydrate isomers that differ in configuration around
Only one specific carbon atom (with the exception of the
Carbonyl carbon) are defined as Epimers of each other.
Ex. Glucose and Galactose are C-4 epimers b/c their structures
differ only in the position of the –OH group at carbon 4.
(Note: the carbons in sugars are numbered beginning at the end
that contains the carbonyl carbon (aldehyde or keto group).
However, b/c galactose and mannose differ in the position of –OH

groups at two carbons ( C-2 & C-4) they are isomers rather than
epimers.
CHO CHO c-2epimerCHO CH2OH
HCOH H C OH HO C C O
HOCH OH C H HOCH HO C H
HO CH H C OH HCOH H C OH
HCOH H C OH HCOH H C OH
CH2OH CH2OH CH2OH CH2OH Isomers
Galactose [c-4 epimers]Glocose Mannose Fructose
B. Enantiomers
A special type of isomerism in w/c the pairs of structures are mirror
images of each other. The presence of asymmetric Carbon atom causes
optical activity.

The two members of the pairs are designated as a D- and an L-sugar. The
vast majority of the sugars in humans are D-sugars. In the isomeric form,

the OH group on the asymmetric carbon (a carbon linked to four

d/t atoms or groups) farthest from the carbonyl carbon is on the
right, whereas in the L-isomer is on the left. Most enzymes are specific
for either the D or the L but enzymes known as recemases are able to
interconvert D- and L- isomers.
C. Cyclization of monosaccharides
Less than 1% of each of the monosaccharides with five or more
carbons exists in open chain form, in solution, rather they are
predominantly found in a ring (Cyclic) form, in w/c the aldehyde
or keto group has reacted with an alcohol group on the same sugar
making the carbonyl carbon asymmetric carbon w/c is called
anomeric carbon.

2. Oligosaccharides

Oligosaccharides contain 2-10 monosaccharide units. The most

abundant oligosaccharides found in nature are the disaccharide.
CARBOHYDRATES
Disaccharides
➢ serve primarily as readily available energy stores.
➢ sucrose, or table sugar, is a major component of plant sap, which
carries chemical energy from one part of the plant to another.
➢Lactose, present in the milk of most mammals, supplies newborn
mammals with fuel for early growth and development.
➢Disaccharides are soluble in water, but they are too big to pass
through the cell membrane by diffusion.

➢ hydrolysis reaction and is the reverse of a condensation reaction and

it releases energy.
➢A condensation reaction takes place by releasing water. This process
requires energy.
CARBOHYDRATES
oligosaccharides
➢ are small chains often found covalently attached to lipids and
proteins, converting them into glycolipids and glycoproteins,
respectively.
➢Oligosaccharides are particularly important on the glycolipids
and glycoproteins of the plasma membrane, where they project
from the cell surface.
➢Because oligosaccharides maybe composed of many different
combinations of sugar units, play an informational role; that is,
they can serve to distinguish one type of cell from another and
help mediate specific interactions of a cell with its surroundings.
Disaccharides
When two monosaccharides are covalently bonded together by
glycosidic bonds a disacharide is formed.
A glycosidic bond is formed when the hydroxy group on one of
the sugars reacts with the anomeric carbon on the second sugar.
Biologically important disaccharides are sucrose, maltose and
lactose
a. Maltose
Contains 2 D-glucose residues joined by a glycosidic linkage b/n
-OH at the C-1 of the first glucose residue and -OH at the C-4
atom of the second glucose forming σ-1,4 glycosidic linkage.
Maltose is the major degradative product of starch. maltose is hydrolyzed
into two σ-glucose by enzyme maltase.
σ-1,4 glycosidic linkage
CH2OH CH2OH

H H OH H H O OH

OH H OH
O H
HO

H OH H OH Structure of Maltose
b. Lactose
Is a disaccharide of β-D-galactose and β-D-glucose w/c are linked by β-1,4-
glycosidic linkage . Lactose acts as a reducing substance since it has a free
carbonyl group on the glucose. It is found exclusively in milk of mammals.
CH2OH CH2O
HO O H O OH
H H O H
H OH H OH H H

H OH H OH β-1,4 -glycosidic bond

c. Sucrose (Cane sugar)
Is a disaccharide of σ-D-glucose and β-D-fructose. It is obtained
from cane sugar and present in various fruits. In contrast to others
sucrose contains no free anomeric C-atom. Since the anomeric
carbons of both its component monosaccharide units are linked to
each other. For this reason sucrose is non reducing sugar.

CH2OH CH2OH

H O O OH
H H
H OH

OH HO H O CH2OH

H OH OH H
3. Polysaccharides
Is the form of high molecular polymers. Are two types in nature:
Homoplysaccaride: that contain only one type of monosaccharide
building blocks
Hetropolysaccharide: w/c contains 2 or more d/t kinds of
monosacharide building blocks
I. Homoplysaccaride
Starch, glycogens, cellulose and dextrins are naturally occurring.
a. Starch
▪ Important storage in plant cell, abundant in tubers, and cereals
▪ Consists of two polymeric units made of glucose called Amylose and
Amylopectin but they differ in molecular architecture.
▪ Amylose is unbranched with 250-300 glucose units linked by σ-1,4
glycosidic linkage
▪ Amylopectin consists of long branched glucose residue with higher
molecular weight.
The inner part of glucose units in amylopectin are joined by σ-1,4
glycosidic linkage as in amylose, but the branch points of
amylopectin are σ-1,6-glycosidic linkages. The branch points reapet
about every 20-30 (1-4) linkages
b. Glycogen
Is the main storage of animal cell, present in liver &
skeletal muscle. Like amylopectin glycogen is a branched
polysaccharide of D-glucose units in σ-1,4 glycosidic linkage , but
is highly branched.
The branches are formed by σ-1,6-glycosidic linkages that occurs
after every 8-12 residue. Therefore liver cell can store glycogen
within a small space. Multiple terminals of branch points release
many glucose units in short time.
c. Cellulose
is the most abundant structural polysaccharide in plants. It is
fibrous, tough, water insoluble.

Is a linear un-branched of 10,000or more D-glucose units connected

by β-1,4 -glycosidic bond.

d. Dextrins
are highly branched of glucose units with σ-1,6, σ-1,4 & σ-1,3
linkages. Since they don’t easily go out of vascular compartment
they are used for intra-venous infusion as plasma volume expander
in the treatment of hypo-volumic shock.
II. Hetro-polysaccharides
Containing more than one type of sugar residues.
a. Glycosaminoglycans, (GAGs) or mucopolysaccharides
They are long, usually un-branched composed of a reapeting
disaccharide units.
The amino sugar is either D- glucosamine or D-galactosamine in w/c
the amino group is usually acetylated, thus eliminating its positive
charge also be sulfated on carbon 4, 6 or on a mono acetylated nitrogen.
The acidic sugar is either D-glucuronic acid or its carbon 6 epimer, L-
uronic acid.
Ex. Hayluronic acid, Heparin and chondatin sulphate.
Functions of Glycosaminoglycans, (GAGs)
➢ they have the special ability to bind large amounts of water, thereby
producing the gel-like matrix that forms the basis of the body’s ground
substance.
➢ since they are negatively charged in bones attract & tightly bind cations
like Ca++, Na+, and K+
➢ Stabilize & support cellular & Fibrous components of tissue while helping
maintain the water & salt balance of the body
➢ essential components of the extra-cellular matrix, & play an important role
in mediating cell-cell interaction.
➢ ground substrate is a part of connective tissue, w/c is a gel like substance
containing water, salt, proteins and polysaccharides. (Ex. Synovial fluid,
w/c serves as a lubricant in joints, and tendon sheaths.
b. Heparin
▪Contains a repeating unit of D-glucuronic and D-gluconsamine, with
sulfate groups on some of the hydroxyl and amine groups.

▪It is an important anticoagulant, prevents the clotting of blood by

initiating the conversion of enzyme prothrombin to thrombin that act on
the conversion of plasma fibrinogen into the fibrin.

▪It is also found in mast cells in lung, liver skin and intestinal mucosa.

c. Glcoproteins (Mucoproteins)

Are proteins to w/c oligosaccharides are covalently attached. They differ

from the glycosaminoglycans in that the length of the glycoproteins
carbohydrate chain is relatively short (2-10).
The glycoprotein carbohydrate chains are often branched instead
of linear and may or may not be negatively charged.

Ex. Glycophorin, found in human red cell membrane

Mucin, a human gastric glycoprotein

many hormones, receptors are glycoprotein

d. Proteoglycans

When glycosaminoglycans are attached to a protein molecule the

compound is called proteoglycan.
CARBOHYDRATES . . .

➢Glycogen_ serves as a storehouse of surplus chemical

energy in most animals.
➢Human skeletal muscles, for example, typically contain
enough glycogen to fuel about 30 minutes of moderate
activity.
➢When stored in cells, glycogen is highly concentrated in
what appears as dark-staining, irregular granules in
electron micrographs
CARBOHYDRATES . . .
➢Most plants bank their surplus chemical energy in the form of
starch, which like glycogen is also a polymer of glucose.
➢Potatoes and cereals, for example, consist primarily of starch.
Starch is actually a mixture of two different polymers, amylose
and amylopectin.
➢Amylose is an unbranched, helical molecule whose sugars are
joined by (1 → 4) linkages, whereas amylopectin is branched.
➢Amylopectin differs from glycogen in being much less branched
and having an irregular branching pattern.
➢Starch is stored as densely packed granules, or starch grains in
plant cells. Although animals don’t synthesize starch, they possess
an enzyme (amylase) that readily hydrolyzes it.
CARBOHYDRATES

➢Cellulose, Chitin, and Glycosaminoglycans: Structural

Polysaccharides Whereas some polysaccharides
constitute easily digested energy stores, others form
tough, durable structural materials.
➢Cotton and linen, for example, consist largely of
cellulose, which is the major component of plant cell
walls.
➢Like glycogen and starch, cellulose consists solely of
glucose monomers; its properties differ dramatically from
these other polysaccharides because the glucose units are
joined by (1 → 4) linkages (bond 3 in rather than (1 → 4)
linkages.
 CARBOHYDRATES

 Chitin is an unbranched polymer of the sugar N acetylglucosamine,

which is similar in structure to glucose but has an acetyl amino group
instead of a hydroxyl group bonded to the second carbon atom of the
ring.
➢Chitin occurs widely as a structural material among invertebrates,
particularly in the outer covering of insects, spiders, and crustaceans.
Chitin is a tough, resilient, yet flexible material not unlike certain
plastics. Insects owe much of their success to this highly adaptive
polysaccharide
6. LIPIDS

➢Lipids are a highly variable group of molecules that

include fats, oils, waxes and some steroids.
➢They are esters of fatty acids and alcohol (glycerol or
chains of alcohols).
➢Fatty acids are made mostly from chains of carbon and
hydrogen and they bond to a range of other types of
atoms to form many different lipids.
➢The primary function of lipids is to store energy.
 LIPIDS

Fats consist of a glycerol molecule linked by ester bonds to three fatty

acids; the composite molecule is termed a triacylglycerol
the structure of fatty acids
➢Fatty acids are long, unbranched hydrocarbon chains with a single
carboxyl group at one end. Because the two ends of a fatty acid
molecule have a very different structure, they also have different
properties.
➢The hydrocarbon chain is hydrophobic, whereas the carboxyl
group (—COOH), which bears a negative charge at physiological
pH, is hydrophilic.
➢Molecules having both hydrophobic and hydrophilic regions are said
to be amphipathic; such molecules have unusual and biologically
important properties.
LIPIDS

➢Fatty acids differ from one another in the length

of their hydrocarbon chain and the presence or
absence of double bonds.
➢Fatty acids present in cells typically vary in
length from 14 to 20 carbons.
➢Fatty acids that lack double bonds, such as
stearic acid are described as saturated; those
possessing double bonds are unsaturated.
➢Naturally occurring fatty acids have double
bonds in the cis configuration. Double bonds (of
the cis configuration) produce kinks in a fatty
acid chain.
LIPIDS
➢Tristearate, whose fatty acids lack double bond, is a
common component of animal fats and remains in a solid
state well above room temperature.
➢In contrast, the profusion of double bonds in vegetable
fats accounts for their liquid state—both in the plant cell
and on the grocery shelf—and for their being labeled as
“polyunsaturated.” Fats that are liquid at room
temperature are described as oils.
➢Solid shortenings, such as margarine, are formed from
unsaturated vegetable oils by chemically reducing the
double bonds with hydrogen atoms (a process termed
hydrogenation).
LIPIDS

➢A molecule of fat can contain three identical fatty acids,

or it can be a mixed fat, containing more than one fatty
acid species.
➢ Most natural fats, such as olive oil or butterfat, are
mixtures of molecules having different fatty acid species.
➢Fats are very rich in chemical energy; a gram of fat
contains over twice the energy content of a gram of
carbohydrate.
➢Carbohydrates function primarily as a short-term, rapidly
available energy source, whereas fat reserves store energy
on a long-term basis.
LIPIDS

Steroids
➢Steroids are built around a characteristic four-ringed
hydrocarbon skeleton.
➢One of the most important steroids is cholesterol, a
component of animal cell membranes and a precursor for
the synthesis of a number of steroid hormones, such as
testosterone, progesterone, and oestrogen.
➢Cholesterol is largely absent from plant cells, which is
why vegetable oils are considered “cholesterol-free,” but
plant cells may contain large quantities of related
compounds.
The structure of steroids
LIPIDS

Phospholipids
➢Phospholipids function primarily in cell membranes
➢The molecule resembles a fat (triacylglycerol), but has only two
fatty acid chains rather than three; it is a diacylglycerol.
➢The third hydroxyl of the glycerol backbone is covalently bonded
to a phosphate group, which in turn is covalently bonded to a small
polar group, such as choline.
➢Thus, unlike fat molecules, phospholipids contain two ends that
have very different properties:
 the end containing the phosphate group has a distinctly hydrophilic
character;
 the other end composed of the two fatty acid tails has a distinctly
hydrophobic character.
LIPIDS
The phospholipid phosphatidylcholine
LIPIDS

Phospholipids
➢Phospholipids function primarily in cell membranes
➢The molecule resembles a fat (triacylglycerol), but has only two
fatty acid chains rather than three; it is a diacylglycerol.
➢The third hydroxyl of the glycerol backbone is covalently bonded
to a phosphate group, which in turn is covalently bonded to a small
polar group, such as choline.
➢Thus, unlike fat molecules, phospholipids contain two ends that
have very different properties:
 the end containing the phosphate group has a distinctly hydrophilic
character;
 the other end composed of the two fatty acid tails has a distinctly
hydrophobic character.
LIPIDS

Properties of lipids
• Insoluble in water
• Longer chains
✓ More hydrophobic, less soluble
• Double bonds increase solubility
• Melting points:
✓ Depend on chain length and saturation
✓ Double bonds lead acyl chain disorder and low melting
temperatures
✓ Unsaturated fatty acids are solid at room temperature.
6. LIPIDS
✓ are a heterogeneous group of water-insoluble organic molecules.
✓ b/c of their insolubility in aqueous solutions:
▪ body lipids are generally found compartmentalized, as in the case
of membrane-associated lipids or droplets of triacylglycerol in
adipocytes,
▪ Transported in plasma in association with protein, as in lipoprotein particles
✓ They are present in all living organisms.
✓The group includes:-
fats, oils, waxes and related compounds.
 General Functions of Lipids
❖ Efficient energy source
❖ Serve as thermal insulator

❖ They are structural components of the cell membrane

❖ Serve as precursors for steroid hormones, prostaglandin that

control the body's homeostasis.

❖ Some fat-soluble vitamins have regulatory or coenzyme

functions.
 5.1 LIPID CLASSIFICATION
There are two ways of classification lipids:

➢ Classification as storage , structural & some other functional lipids

➢ Classification based on Lipid composition

1. Simple lipids

Is esters of fatty acids with d/t alcohols, fats & oils are esters of fatty
acids with glycerol

Waxes: Esters of fatty acids with high molecular weight monohydric

alcohols

2. Complex lipids

Esters of fatty acids & alcohols together with some other head groups
a. Phospholipids:
Esters of the above type containing phosphoric acid residue

ex. Glycerophospholipds: the alcohol is glycerol

Sphingophospholipids: the alcohol is sphingosine

b. Glycolipids: Lipids containing fatty acids, sphingosine and

carbohydrate residues.
H2C OH
HC OH
H2C OH
Glycerol
Others: include sulfolipids, amino lipids & lipoproteins w/c are
modified form of lipids.

3. Derived lipids:

Include the hydrolytic products of the simple and complex lipids.

Ex. Fatty acids, cholesterol….

The simplest naturally occurring lipids are triacylglycerols formed

by esterification of fatty acids with glycerol.

Biological membranes are made up of phospholipids, glycolipids

and proteoglycans.
 a. FATTY ACIDS
▪ are building blocks of most lipid, made from a carboxylic acids
(head) with long-chain hydrocarbon side groups (tail).
▪They are rarely free in nature but, rather, occur in esterified forms.
▪ Most naturally occurring fatty acids have an even number of carbons.
▪They may be saturated or unsaturated with one or more double bonds.
Mostly the double bond occurs at the 9th carbon as we count from the
carboxyl group end.

▪In higher plants and animals, the predominant fatty acid residues are
those of the C-16 and C-18 species palmitic, oleic, linoleic, and
stearic acids. Fatty acids with 14 or 20 carbon atoms are
uncommon.
▪ Over half of the fatty acid residues of plant and animal lipids are unsaturated
(contain double bonds) and are often polyunsaturated (contain two or more
double bonds).

▪ Bacterial fatty acids are rarely polyunsaturated but are commonly branched,
hydroxylated, or contain cyclopropane rings.

▪ Unusual fatty acids also occur as components of the oils and waxes (esters
of fatty acids and long-chain alcohols) produced by certain plants.

Ex. CH3 (CH2)7 CH2CH2 (CH2)7 COOH stearic acid (saturated fatty acid)

CH3 (CH2)7 CH CH2 (CH2)7 COOH Oleic acid ( Unsaturated fatty acid)

Fatty acids can be represented as C18:1, Δ9 where Δ indicates the position of the
double bond b/n (9th & 10th ) C-atom , 18 is the number of C-atom, 1 is the
number of double bonds.
Double bonds in naturally occurring fatty acids are in Cis-configuration
and saturated fatty acids of C12 – C24 are solids at physiologic temp. but
the unsaturated once are liquids.
Poly unsaturated fatty acids ( PUFA): have two or more double
bonds; they are called as essential fatty acids b/c they are required in the
body and can not be synthesized so need to included in the diet.
Ex. Linoleic acid 18:2: Δ(9,12)
Linolenic acid 18:3: Δ(9,12, 15) Essential fatty acids
Arachidonic acid 20:4: Δ(5,8, 11, 14) semi-essential b/c synthesized
from the above two
Function of PUFA
✓ required for the synthesis of phospholipids, cholesterol, ester & lipoproteins
✓ PFA are released from membrane, diverted for the synthesis of
prostaglandin, leukotreins and thromboxanes.
✓Act as mobilizing agents in liver & protect liver from accumulating fats
The common biological fatty acids
Saturated fatty acids

Symbol Common Name Systemic name structure MP (Co )

12 Lauric acid Dodecanoic acid CH3(CH2)10COOH 42.2

14 Myristic acid Tetradecanoic acid CH3(CH2)12COOH 52
16 Palmitic acid Hexadecanoic acid CH3(CH2)14COOH 63.1
18 Stearic acid Octadecanoic acid CH3(CH2)16COOH 69.6
20 Arachidic acid Eicosanoic acid CH3(CH2)18COOH 75.4
22 Arachidic acid Docosanoic acid CH3(CH2)20COOH 81
24 Lignoceric acid Tetracosanoic acid CH3(CH2)22COOH 84.2
 Unsaturated fatty acids
Symbol Common Name Systemic name Structure
(Co)
16:1n–7 Palmitoleic acid 9-Hexadecenoic acid CH3(CH2)5CH CH(CH2)7COOH -0.5
18:1n–9 Oleic acid 9-Octadecenoic acid CH3(CH2)7CHCH(CH2)7COOH 13.4

18:2n–6 Linoleic acid 9,12-Octadecadienoic CH3(CH2)4(CH CHCH2)2(CH2)6COOH -9

acid
18:3n–3 α-Linolenic acid 9,12,15- CH3CH2(CH CHCH2)3(CH2)6COOH -17
Octadecatrienoic acid
18:3n–6 γ-Linolenic acid 6,9,12- CH3(CH2)4(CH CHCH2)3(CH2)3COOH
Octadecatrienoic acid
20:4n–4 Arachidonic acid 5,8,11,14- CH3(CH2)4(CH CHCH2)4(CH2)2COOH -49.5

Eicosatetraenoic acid
20:5n–3 EPA 5,8,11,14,17- CH3CH2(CH CHCH2)5(CH2)2COOH -54
Eicosapentaenoic acid
22:6n–3 DHA 4,7,10,13,16,19- CH3CH2(CH CHCH)6CH2COOH
Docosahexenoic acid
24:1n–9 Nervonic acid 15-Tetracosenoic acid CH3(CH2)7CH CH(CH2)13COOH 39
 b. TRIACYLGLYCEROL
The fats and oils that occur in plants and animals consist largely of
mixtures of triacylglycerols (also referred to as triglycerides or
neutral fats). These nonpolar, water-insoluble substances are fatty
acid triesters of glycerol:
H2C OH O
HC OH H2C O C R1
H2C OH O
Glycerol HC O C R2 triacylglycerol
O
H2C O C R3
✓ Triacylglycerols function as: energy reservoirs in animals and are
therefore their most abundant class of lipids even though they are not
components of biological membranes
✓ Tristerearin is a chief component of beaf lipid
✓ Butter has a short chain fatty acid
✓ Unsaturated fatty acids are sensitive to air and oxidized to give
rancid smell
✓ Triacylglycerol is mainly found in special cells called adipocytes of the
mammary gland, abdomen and under skin of animals
Triacylglycerols differ according to the identity and placement of their three
fatty acid residues. The so-called simple triacylglycerols contain one type of
fatty acid residue and are named accordingly. For example,
tristearoylglycerol or tristearin contains three stearic acid residues,
whereas trioleoylglycerol or triolein has three oleic acid residues. The
more common mixed triacylglycerols contain two or three different types of
fatty acid residues and are named according to their placement on the glycerol
moiety.
H2C O C1 (CH2)7 CH CH (CH2)5 CH3

HC O C2 (CH2)7 CH CH CH2 CH CH (CH2)4 CH3

H2C O C3 CH2 (CH2)7 CH2 CH2 (CH2)6 CH3

1-Palmitoleoyl-2-linoleoyl- 3-stearoylglycerol
Fats and oils (which differ only in that fats are solid and oils are liquid
at room temperature) are complex mixtures of simple and mixed
triacylglycerols whose fatty acid compositions vary with the organism
that produced them. Plant oils are usually richer in unsaturated fatty
acid residues than are animal fats, as the lower melting points of oils
imply.
 Triacylglycerols are Efficient Energy Reserves
❖ Fats are a highly efficient form in which to store metabolic energy.
This is because fats are less oxidized than are carbohydrates or
proteins and hence yield significantly more energy on oxidation.

❖ In animals, adipocytes (fat cells) are specialized for the synthesis

and storage of triacylglycerols. Whereas other types of cells have only
a few small droplets of fat dispersed in their cytosol, adipocytes may
be almost entirely filled with fat globules.

❖ Adipose tissue is most abundant in a subcutaneous layer and in

the abdominal cavity
▪ The fat content of normal humans (21% for men, 26% for women)
enables them to survive starvation for 2 to 3 months.

▪ In contrast, the body’s glycogen supply, which functions as a short-

term energy store, can provide for the body’s metabolic needs for less
than a day.

▪ The subcutaneous fat layer also provides thermal insulation, which is

particularly important for warm-blooded aquatic animals, such as
whales, seals, geese, and penguins, which are routinely exposed to low
temperatures.
 C. Glycerophospholipids
Glycerophospholipids (or phosphoglycerides) are the major lipid
components of biological membranes. They consist of sn-glycerol-3-
phosphate esterified at its C1 and C2 positions to fatty acids and at its
phosphoryl group to a group, X, to form the class of substances.
Glycerophospholipids are therefore amphiphilic molecules with non-
polar aliphatic “tails” and polar phosphoryl-X “heads.” The simplest
glycerophospholipids, in which X = H, are phosphatidic acids; they
are present only in small amounts in biological membranes. In the
glycerophospholipids that commonly occur in biological membranes,
the head groups are derived from polar alcohols.
 CH2 OH
HO C H O O
CH2 O P OH O CH2 O C R1
OH R2 C O C H O
sn-Glycerol-3-phosphate CH2 OP O X
O-
Glycerophospholipid
The Common Classes of Glycerophospholipids:
If the above ‘X’ is H the name of phospholipid be Phosphatidic acid
CH2CH2NH3+ (Ethanolamine) is Phosphatidylethanolamine
CH2CH2N(CH3)3+ (Choline) is Phosphatidylcholine (lecithin)
CH2CH(NH3 )COO- (Serine) is phosphatidylserine
CH2CH(OH)CH2OH (Glycerol) is Phosphatidylglycerol
Saturated C16 and C18 fatty acids usually occur at the C1 position of
glycerophospholipids, and the C2 position is often occupied by an
unsaturated C16 to C20 fatty acid. Glycerophospholipids are, of course,
also named according to the identities of these fatty acid residues . Some
glycerophospholipids have common names. Ex. phosphatidylcholines
are known as lecithins; diphosphatidylglycerols, the “double” glycerol
phospholipids, are known as cardiolipins (because they were first
isolated from heart muscle).
 D. Sphingolipids
Sphingolipids, which are also major membrane components, are
derivatives of the C18 amino alcohols sphingosine, dihydrosphingosine
Their N-acyl fatty acid derivatives, occur only in small amounts in
plant and animal tissues but form the parent compounds of more
abundant sphingolipids:
OH H OH OH H OH
H2C C C H H2C C C H
H3N+ CH H3N+ CH2
HC CH2
(CH2)12 CH2)12
CH3 CH3
Sphingosine Dihydrosphingosine
 E. Cholesterol
Steroids, which are mostly of eukaryotic origin, are derivatives of
cyclopentanoperhydrophenanthrene .

▪ The most abundant steroid in animals, is further classified as a sterol b/c

of its C3-OH group and its branched aliphatic side chain of 8 to 10 carbon
atoms at C17. Its polar OH group gives it a weak amphiphilic character,
whereas its fused ring system provides it with greater rigidity than other
membrane lipids.
➢ Cholesterol is a major component of animal plasma membranes,
therefore an important determinant of membrane properties.
➢It is also abundant in blood plasma lipoproteins, where 70% of it is
esterified to long-chain fatty acids to form cholesteryl esters.
➢ Cholesterol is the metabolic precursor of steroid hormones, substances
that regulate a great variety of physiological functions including sexual
development and carbohydrate metabolism.
➢ for the synthesis of bile salts that are important in lipid digestion &
absorption
➢ for the synthesis of vitamin D
➢ As a component of lipoproteins as transport forms of lipid based
energy
Plants contain little cholesterol. Rather, the most common sterol components of
their membranes are stigmasterol and sitosterol, which differ from
cholesterol only in their aliphatic side chains. Yeast and fungi have yet
other membrane sterols such as ergosterol, which has a C7 to C8 double
bond.
NUCLEIC ACIDS

➢Nucleic acids are complex macromolecules that store and transmit genetic
information
➢Nucleic acids are made of smaller repeating subunits called nucleotides
➢Nucleotides are composed of carbon, nitrogen, oxygen, phosphorus, and
hydrogen atoms
➢Function:
➢store & transmit hereditary information
➢Two Nucleic Acids:
➢RNA (ribonucleic acid)
➢DNA (deoxyribonucleic acid)
➢Structure:
➢monomers = nucleotides
There are five (not six) major nucleotides, all of which
have three units a phosphate, anitrogenous base, and
a ribose sugar
 3 parts
NUCLEOTIDES
 nitrogen base (C-N ring)
 pentose sugar (5C)
 ribose in RNA
 deoxyribose in DNA
 PO4 group
 TYPES OF NUCLEOTIDES
 2 types of nucleotides
 different Nitrogen bases
 purines
 double ring N base
 adenine (A)
 guanine (G)
 pyrimidines
 single ring N base
 cytosine (C)
 thymine (T)
 uracil (U)
BUILDING THE POLYMER _ NUCLEOTIDES
 NUCLEIC ACID
POLYMERIZATIONS
➢Backbone
➢sugar to PO4 bond
➢phosphodiester bond
➢ new base added to sugar of
previous base
➢ polymer grows in one
direction
➢N bases hang off the
sugar-phosphate backbone
 RNA & DNA

➢RNA
➢single nucleotide chain
➢DNA
➢double nucleotide chain
➢ N bases bond in pairs
across chains

➢spiraled in a double helix

➢ double helix 1st proposed as structure of
DNA in 1953 by James Watson & Francis
Crick
 PAIRING OF NUCLEOTIDES

➢Nucleotides bond between

DNA strands
➢H bonds
➢purine :: pyrimidine
➢A :: T
➢ 2 H bonds
➢G :: C
➢ 3 H bonds
❖ DNA is soluble in water.

❖ DNA is insoluble in ethanol or purified alcohol

❖ DNA absorbs ultraviolet light.

❖ DNA can be denatured and re-natured.

Structure of the DNA
❖ Two long strands make the shape of a double helix.

❖ Two strands run in opposite directions to each other and are

therefore anti-parallel.

❖ Chemically, DNA consists of two long polymers of simple units

called nucleotides, with backbones made of base, sugars and
phosphate groups. 170
❖ The DNA double helix is stabilized both by hydrogen bonds
between the bases.

Watson and Crick’s Structural Model of DNA

❖ Two helical polynucleotide chains are coiled around a common

axis. The chains run in opposite directions.

❖ The sugar-phosphate backbones are on the outside and, therefore,

the purine and pyrimidine bases lie on the inside of the helix.

❖ Adjacent bases are separated by 0.34 Å. The helical structure

repeats every 3.4 Å, so there are 10 bases ( 34 Å per repeat/3.4 Å
per base) per turn of helix.
171
❖ The diameter of the helix is 20 Å.

❖ Thus, the two chains are complementary to each other.

❖ The two strands run anti-parallely that is, have opposite directions.

❖ One strand has phosphodiester linkage in 3'→ 5' direction, while

other strand has phosphodiester linkage in just reverse or 5'→3'
direction.

❖ The helix has two external grooves, a deep wide one, called major
groove and a shallow narrow one, called minor groove

❖ Both of these grooves are large enough to allow protein molecules

to come in contact with the bases.
172
173
Alternative Forms of DNA Double Helices

• There are three fundamental types of double helix structure of

DNA such as: B, A, and Z-DNA.

1. A-DNA
➢ Is right-handed helix.

➢ The major groove of A-DNA is deep and narrow, while the minor
groove is shallow and broad.

2. Z-DNA
➢ Formed a left-handed helix.

➢ The minor groove is very deep and narrow and the major groove is
175
shallow to the point of being virtually nonexistent.
3. B-DNA
➢ B-DNA is a right-handed helix; it turns in a clockwise manner.

➢ The major groove is wide and of moderate depth, while the minor
groove is of moderate depth but is much narrower.

176
177
Chargaff Rule

• Chargaff, in 1950, discovered the equivalence rule which suggested

that despite wide compositional variations exhibited by different
types of DNA, the total amount of purines equaled the total amount
of pyrimidines (A+G=T+C);

• The amount of adenine equaled the amount of thymine (A=T) and

• The amount of guanine equaled the amount of cytosine (G=C).

• Chargaff’s equivalence rule has been found to apply almost

universally in different organisms.

178
1. Assume the following base sequence was found in a 20 base DNA
strand : 3' ATT CGA CCT TAT TAC TGC AC 5'

A. What would be the first 5 bases in the 3´ and 5' end of the
complementary strand?

B. Assuming the presence of complementary strands, what is the per

cent composition of the polymer with respect to AT base pairs
and with respect to GC base pairs.

2. Analysis of four double-stranded DNA samples yielded the

following information:

• 5% cytosine ; 12% guanine; 28% thymine ; 60% adenine. What

would be the percentage of the other bases in each sample?
The Central Dogma of Cell

• Genetic information flows from DNA to RNA to protein during

cell growth.

• In addition, all living cells must replicate their DNA when they
divide.

• The central dogma of molecular biology is a scheme showing the

flow of genetic information during both the growth and division of
a living cell.

• During cell division each daughter cell receives a copy of the

genome of parent cell. As the genome is present in the form of
DNA, cell division involves the duplication of this DNA
180
 REPLICATION •DNA
(DNA)

mRNA
TRANSCRIPTION •RNA
(RNA)

tRNA
TRANSLATION
(PROTEIN)
• PROTEIN

181
 2.1.2 The molecular basis of DNA replication
❖ A process of making number of copies of DNA molecule is
called replication.

DNA Replication Models

1. Semi-conservative: Two daughter duplexes with one each of

the old and new strands.

2. Conservative: Old duplex is conserved and new duplex

composed of two completely new strands.

3. Dispersive: Sections of the old duplex dispersed somewhat

randomly to the two daughter duplexes.
182
183
❖ Meselson and Stahl’s experiment verified the
semiconservative nature of DNA replication in a series of
elegant experiments using isotopically labelled DNA and a
form of isopycnic density gradient centrifugation.

❖ They grew Escherichia coli in medium containing 15N, a

heavy isotope of nitrogen.

❖ After many generations they transferred the bacteria with

heavy (15N) DNA to a medium containing only 14N.

❖ Two bands at the first generation of replication an original

15N (heavy) double helix and a new 14N (light) double helix

184
185
186
Modes of Replication

✓ Individual units of replication are called replicons, each of

which contains a replication origin.

✓ Replication starts at the origin and continues until the entire

replicon has been replicated.

✓ The point of unwinding, where the two single nucleotide

strands separate from the double-stranded DNA helix, is called
a replication fork.

✓ If there are two replication forks, one at each end of the

replication bubble, the forks proceed outward in both
directions in a process called bidirectional replication.
187
Requirements of Replication

1. A template consisting of single-stranded DNA,

2. Raw materials (substrates) to be assembled into a new nucleotide

strand, and

3. Enzymes and other proteins that “read” the template and assemble
the substrates into a DNA molecule.

Direction of Replication

✓ All DNA synthesis is 5’ to 3’, new nucleotides are always added to

the 3’ end of the growing nucleotide strand.

✓ DNA polymerases, the enzymes that synthesize DNA, can add

nucleotides only to the 3’ end of the growing strand. 188
✓ As the DNA unwinds, the template strand that is exposed in the
3’ to 5’ direction allows the new strand to be synthesized
continuously, in the 5 to 3’ direction.

✓ This new strand, which undergoes continuous replication, is

called the leading strand.

✓ The newly made strand that undergoes discontinuous replication

is called the lagging strand.

✓ The short lengths of DNA produced by discontinuous replication

of the lagging strand are called Okazaki fragments.

✓ If the bubble has two forks, one at each end, synthesis takes place
simultaneously at both forks (bidirectional replication).189
190
Figure: DNA synthesis takes place simultaneously but in opposite
directions on the two DNA template strands. 191
192
Enzymes involved in DNA replication

193
 The process of DNA replication in prokaryotes

❖ Replication takes place in three stages: initiation,

elongation, and termination.

1. Initiation

❖ DNA replication begins at specific sites on the DNA

molecule, called origins of replication.

❖ The circular chromosome of E. coli has a single replication

origin (oriC).

❖ Replication is initiated at a replication origin, where an

initiator protein binds and causes a short stretch of DNA to
unwind. 194
✓ Then DNA helicase breaks hydrogen bonds at a replication
fork, and single-strand-binding proteins stabilize the separated
strands.

✓ DNA gyrase reduces torsional strain that develops as the two

strands of double helical DNA unwind or prevent supercoiling.

✓ Primers: All DNA polymerases require a nucleotide with a 3’-

OH group to which a new nucleotide can be added.

✓ An enzyme Primase synthesizes a short stretch of RNA

nucleotides called primers (about 10–12 nucleotides long),
which provides a 3’-OH group for the attachment of DNA
nucleotides to start DNA synthesis.
195
✓ All DNA molecules initially have short RNA primers
imbedded within them; these primers are later removed and
replaced by DNA nucleotides.

✓ On the leading strand, where DNA synthesis is continuous, a

primer is required only at the 5’ end of the newly synthesized
strand.

✓ On the lagging strand, where replication is discontinuous, a

new primer must be generated at the beginning of each
Okazaki fragment.

✓ Primase forms a complex with helicase at the replication fork

and moves along the template of the lagging strand.
196
197
198
2. Elongation

➢ After DNA is unwound and a primer has been added, DNA

polymerases elongate the polynucleotide strand by catalyzing
DNA polymerization.

➢ DNA polymerase add nucleotide at 3’ end.

➢ There are least five different types of DNA polymerases.

➢ Two of them, DNA polymerase I and DNA polymerase III,

carry out DNA synthesis associated with replication; the other
three have specialized functions in DNA repair.

➢ DNA polymerase III is a large multi-protein complex that acts

as the main workhorse of replication. 199
➢ DNA polymerase III synthesizes nucleotide strands by adding
new nucleotides to the 3’ end of growing DNA molecules.
➢ This enzyme has two enzymatic activities:
➢ Its 5’ 3’ polymerase activity allows it to add new
nucleotides in the 5’ 3’ direction.
➢ Its 3’ 5’ exonuclease activity allows it to remove
nucleotides in the 3’ 5’ direction, enabling it to correct
errors.
➢ If a nucleotide having an incorrect base is Inserted into the
growing DNA molecule, DNA polymerase III uses its 3’ to 5’
exonuclease activity to back up and remove the incorrect
nucleotide. 200
➢ It then resumes its 5’ 3’ polymerase activity.
➢ These two functions together allow DNA polymerase III to
efficiently and accurately synthesize new DNA molecules.
➢ DNA polymerase I, also has 5’ 3’ polymerase and 3’ 5’
exonuclease activities permitting the enzyme to synthesize
DNA and to correct errors.
➢ Unlike DNA polymerase III, however, DNA polymerase I also
possesses 5’ 3’ exonuclease activity, which is used to
remove the primers laid down by primase and to replace them
with DNA nucleotides by moving in a 5’ 3’ direction.
➢ The removal and replacement of primers appear to constitute
the main function of DNA polymerase I. 201
Table

202
➢ After primers are removed and replaced, the nick in the sugar–
phosphate linkage is sealed by DNA ligase.

3. Termination
➢ In some DNA molecules, replication is terminated whenever
two replication forks meet.

➢ In others, specific termination sequences block further

replication.

➢ A termination protein, called Tus in E. coli, binds to these

sequences.

➢ Tus blocks the movement of helicase, thus stalling the

replication fork and preventing further DNA replication.
203
204
205
206