THE UNIVERSITY OF ZAMBIA

SCHOOL OF MEDICINE

DEPARTMENT OF PHYSIOLOGICAL SCIENCES

Telephone: 252641 P.O. Box 50110
211440 (UTH) 254824 (Pre-Clinical) Ridgeway Campus Lusaka, Zambia
Fax: + 260 – 1 – 250753

Bachelor of Science in Nursing

BIOCHEMISTRY BCN 215

MODULE 4 (Metabolism 2)
(Fatty acid metabolism, cholesterol metabolism, amino acid metabolism, nucleic acid
metabolism and protein synthesis)

Gibson Sijumbila

BSc (Human Biology) University of Zambia

MB ChB, University of Zambia

MD, University of Dundee, UK

First Edition - 2011

1
Index
Page number
Introduction to module 4……………………………………………………………………….. 4
Aim…………………………………………………………………………………………. 4
Objectives…………………………………………………………………………………….. 4
Study skills…………………………………………………………………………………… 5
Need help…………………………………………………………………………………….. 5
Time frame…………………………………………………………………………………… 5
Structure of module………………………………………………………………………….. 5
Assessment ………………………………………………………………………………….. 5
Prescribed Readings………………………………………………………………………….. 5
Recommended Readings……………………………………………………………………... 5
Unit 1 Fatty acid metabolism………………………………………………………………...... 7
Introduction…………………………………………………………………………………… 7
Aim ………………………………………………………………………………………….. 7
Objectives…………………………………………………………………………………….. 7
Equipment …………………………………………………………………………………… 7
Other resources……………………………………………………………………………….. 7
Content……………………………………………………………………………………….. 7
Lipid digestion and absorption………………………………………………………………… 7
Fatty acid oxidation…………………………………………………………………………….. 9
Biosynthesis of fatty acids……………………………………………………………………… 12
Source of NADPH for fatty acid synthesis……………………………………………………. 14
Short term and long term regulation of fatty acid synthesis……………………………….. 15
Questions…………………………………………………………………………………………. 15
Unit 2 Cholesterol metabolism…………………………………………………………………. 16
Introduction…………………………………………………………………………………… 16
Aim…………………………………………………………………………………………… 16
Objective……………………………………………………………………………………... 16
Equipment …………………………………………………………………………………… 16
Other resources……………………………………………………………………………….. 16
Content ………………………………………………………………………………………. 17
Biosynthesis of cholesterol…………………………………………………………………….. 17
Regulation of cholesterol synthesis……………………………………………………………. 18
Fate of cholesterol………………………………………………………………………………. 18
Bile acids and salts……………………………………………………………………………… 19
Questions…………………………………………………………………………………………. 20
Unit 3 Amino acid metabolism………………………………………………………………… 21
Introduction…………………………………………………………………………………… 21
Aim…………………………………………………………………………………………… 21
Objective……………………………………………………………………………………... 21
Equipment……………………………………………………………………………………. 21
Other resources……………………………………………………………………………….. 21
Content……………………………………………………………………………………….. 21
Amino acid synthesis……………………………………………………………………………. 21
Catabolism of amino acid nitrogen…………………………………………………………… 24
Urea synthesis……………………………………………………………………………………. 25
Questions…………………………………………………………………………………………. 26
Unit 4Nucleic acid metabolism…………………………………………………………… 28
Introduction…………………………………………………………………………………… 28
Aim…………………………………………………………………………………………… 28
Objective……………………………………………………………………………………... 28
Equipment……………………………………………………………………………………. 28
Other resources……………………………………………………………………………….. 28
Content……………………………………………………………………………………….. 29
DNA synthesis…………………………………………………………………………………… 29
Origin of replication……………………………………………………………………………. 29
Unwinding of DNA……………………………………………………………………………... 29
Formation of replication fork……………………………………………………………… 29
Initiation and elongation……………………………………………………………………… 29
Formation of replication bubbles……………………………………………………………. 29

2
 RNA synthesis…………………………………………………………………………………… 33
Synthesis of DNA from RNA………………………………………………………………….. 35
Questions………………………………………………………………………………………….. 35
Unit 5 Protein synthesis………………………………………………………………………… 33
Introduction…………………………………………………………………………………… 33
Aim…………………………………………………………………………………………… 33
Objective……………………………………………………………………………………... 33
Equipment……………………………………………………………………………………. 33
Other resources……………………………………………………………………………….. 33
Content……………………………………………………………………………………….. 34
Stages of protein synthesis……………………………………………………………………… 34
Antibiotic inhibitors of protein synthesis…………………………………………………….. 36
Questions ………………………………………………………………………………………… 36
Summary of the module…………………………………………………….. 36

*****

3
 MODULE 4

Introduction
This module is a continuation of metabolism with emphasis on fatty acids, cholesterol, amino
acids, nucleic acids and proteins. Fatty acid oxidation, a process which occurs in the
mitoncondria will be discussed first followed by fatty acid synthesis. The role of fatty acid
oxidation in energy generation and the convertion of excess carbohydrates into fatty acids
will are emphasized. Cholesterol, a form of lipid is well known for all the pathological
conditions it causes but the truth is it is a precursor for a number of important molecules like
steroid hormones, bile acids. Knowledge of cholesterol metabolism is vital in order to
understand the pathophysiology of the disease processes linked to abnormal cholesterol
metabolism. Apart from being monomer units of proteins, amino acids have many functions
in the body. For instance amino acids can be a source of energy during starvation and this in
part explains the muscle loss during starvation. Some amino acids are synthesized in the body
(non essential amino acids) and others can not, and therefore need to be taken in diet
(essential amino acids). Abnormalities in metabolism of some amino acids can give rise to
severe clinical conditions. Nucleic acid metabolism is important in cell replication and protein
synthesis. Cell division is preceeded by DNA replication and protein synthesis is preceeded
by RNA replication (transcription). Any mutation or damage to DNA is likely to be translated
into an abnormal protein whose functional capacity depends on the the type and location of
DNA change and this can result in disease. Some important chemotherapeautic drugs work by
inhibiting nucleic acid or protein synthesis. Survival of organisms to a large extent relies on
the capacity of cells to replicate. If DNA synthesis can be selectively inhibited in
microorganisms then the inhibitor would be a useful chemotherapeautic drug against that
microorganism. Similary by inhibiting DNAsynthesis some rapidly growing malignancies can
be controlled.

Aim: The aim is to give you essential knowledge about fatty acid, cholesterol, amino
acid and nucleic acid metabolism and the common diseases that result from their abnormal
metabolism.

Objectives By the end of this module you should be able to:

1. Outline the steps of fatty acid oxidation and fatty acid synthesis
2. Explain how ketone bodies are formed
3. Name important derivatives of cholesterol
4. List essential and non essential amino acids
5. Name diseases that result from abnormal amino acid metabolism
6. Outline the process of DNA replication
7. Explain what genetic code is, and how information is passed on from the nucleus
during protein synthesis.

Study skills.
The notes you will find under the core text are comprehensive enough for you to pass your
exams. However it is strongly recommended that you read the prescribed and recommended

4
text books as well so that you can broaden your understanding of the subjects. Certain
websites have very good information on various topics in biochemistry; so whenever possible,
please check on various websites shown in respective units under ´Other Resources´. Please
note that molecular structures have been included solely for reference purposes. You are not
supposed to memorize molecular structures as that would be beyond the scope of the course.
You are also advised to join small discussion groups if there are other students in the vicinity
doing the same course.

Need help
In case you have difficulties during the course please get in touch with the Director, Institute
of Distance Education, or the resident lecturer in your province.

Time frame
You will be expected to spend at least 60 hours of study time on this module.

Structure of the module.

This module is divided into five units and each of the units has notes you are supposed to
study and at the end of each unit you will find some questions which you will be expected to
answer. The distribution of topics is as follows: Unit 1 fatty acid metabolism, Unit 2
cholesterol metabolism, Unit 3 amino acid metabolism, Unit 4 nucleic acid metabolism and
Unit 5 protein synthesis

Assessments
Continuous assessment 40%
Assignments 10%
Tests 30%
Final 60%

Prescribed Readings:

1. Reginald H. Garrett and Charles M. Grisham (2007). Biochemistry, Third Edition

ISBN-13: 978-0-497-11912-8
2. Michael M. Cox and David L. Nelson (2008). Lehninger Principles of Biochemistry, Fifth
Edition. ISBN:978-0-230-22699-9

Recommended Readings:

1. Jeremy M. Berg, John L. Tymoczko and Lubert Stryer (2006) Biochemistry, Sixth
Edition. ISBN-13: 978-0-7167 – 8724 – 2.
2. John W. Baynes, Marek H. Dominiczak (2008), Medical Biochemistry, Second Edition.
ISBN 978 0 7234 3341 5
3. Jeannette Naish, Patricia Revest and Denise S. Court (2009), Medical Sciences, First
Edition. ISBN: 978 0 702 026 799
4. MN Chatterjea and Rana Shinde (2008), Medical Biochemistry, Seventh Edition. ISBN
81 – 8448 – 134 – 9

5
5. Barth, G Butler, P Hammond. Biochemical investigations in laboratory medicine. 2001,
ACB Venture pubs. ISBN 0-902-42934-5.

6
 UNIT 1
FATFY ACID METABOLISM

1.1 Introduction
Fatty acids have four major functions. They are components of phospholipids and glycolipids
which form lipid bilayers in cell membranes. By attaching to proteins, they target the proteins
to membrane locations or anchor them in the membrane. Fatty acids are also fuel molecules
which are much more efficient than glycogen because they are more highly reduced than
carbohydrates, and do not carry a lot of water unlike carbohydrates.
Fatty acids are stored as fats, or triacylglycerols in adipose tissue. In addition they also act as
intracellular messengers. Mobilization of fatty acids is initiated by hormones (epinephrine,
norepinephrine, glucagon, adrenocorticotropic hormone), and mediated intracellularly by c-
AMP in a manner similar to mobilization of glycogen. Hydrolysis of triglycerides is catalyzed
by a cellular lipase. Any glucose or intermediates of glucose oxidation in excess of body
requirements, and after replenishing glycogen stores is converted into fatty acids which are
stored in adipose tissues as triglycerides.

1.2 Aim
The aim of the unit is to improve your understanding of the role of fatty acids in energy flow
in the body.

1.3 Objectives
By the end of the unit you should be able to:
1. Explain how dietary triglycerides are absorbed.
2. Outline the process of fatty acid oxidation.
3. Out line the process of fatty acid synthesis.
4. Explain the effect of diabetes and starvation on fatty acid metabolism.

1.4 Equipment
None

1.5 Other resources

a) [Link]
b) [Link]
c) [Link]
d) [Link]

1.6 Contents
Lipid Digestion and Absorption.
Dietary lipids are primarily (90%) triacylglycerols but also include cholesterolesters,
phospholipids, essential unsaturated fatty acids and fat-soluble vitamins (A, D, E, K).

7
Essentially nearly all (98%) of the fat consumed in the diet is absorbed, and most is
transported to adipose tissues for storage.
Lingual lipase followed by gastric lipase start minor digestion of triacyglycerols. These
enzymes remove one of the three fatty acids leaving diacylglycerol and free fatty acid as a
product.
triacylglycerol → fatty acid + diacylglycerol (lingual or gastric lipase)
The major digestion of all lipids occurs in the lumen of the small intestines. Pancreatic lipase
removes the fatty acids from carbons one and three of the glycerol backbone. This enzyme
requires for its activity colipase, a small protein. Cholesterol esterase, removes the ester
portion of dietary cholesterol ester to facilitate its uptake across the intestinal wall.
Phospholipase A2 removes a fatty acid from carbon 2 of the glycerol backbone leaving a fatty
acid on carbon 1 and the polar headgroup on carbon 3 (lysophospholipid).
triacylglycerol → 2 fatty acids + monoacylglycerol (pancreatic lipase)

cholesterol ester → cholesterol + ester (cholesterol esterase)

phospholipids → fatty acid + lysophospholipid (phospholipase A2)

Lipids are solubilized by biological detergents, bile salts. Bile acids are formed by the liver
from cholesterol and converted to bile salts by intestinal bacteria. Bile salts act in the
absorption of lipids by reversibly forming micelles. Bile salts form mixed micelles with lipids,
such as 2-monoacylglycerol, phospholipids, fatty acids, cholesterol and fat soluble vitamins.
During triacylglycerol digestion by pancreatic lipase, free fatty acids and monoacylglycerols
are released at the surface of fat emulsion droplets. Products of triacylglycerol hydrolysis are
continuously transferred from emulsion droplets to the mixed micelles.

Uptake of lipids by the epithelial cells lining the intestines occurs by passive diffusion from
the mixed micelles through the membrane. Cholesterol is converted back to its cholesterol
ester form and fatty acids are reattached to glycerol backbones to form new molecules of
triacylglycerol. Lipid products are then incorporated into lipoproteins known as a
chylomicrons which are exported into the lymphatic system and delivered to tissues such as
adipose tissue and muscle. You must also realize that there are several other types of
lipoproteins whose composition and functional details are beyond the scope of the course
(Harper 2003:475).

Summary of the steps of lipid digestion and absorption

1. Minor digestion of triacylglycerols in the mouth and stomach by lingual and gastric
lipases respectively.
2. Major digestion (all lipids) occurs in the lumen of the small intestines and catalyzed by
pancreatic lipase, cholesterol esterase, and phospholipase A2 as described above.
3. Formation of mixed micelles in lumen of the small intestines.
4. Passive absorption of lipolytic products into intestinal epithelial cell.
5. Assembly and export of chylomicrons from intestinal cells to the lymphatics.

8
Fatty acid oxidation (β-Oxidation)
Fatty acids are mobilized from triglycerides by hormone sensitive lipase. The hormones
epinephrine and glucagon through cAMP lead to phosphorylation and activation of hormone
sensitive lipase. The activated hormone then frees the fatty acids from ester linkages with
glycerol. The fatty acids are circulated bound to albumin as free fatty acids to tissues where
they are to be oxidized for energy.
Fatty acid oxidation occurs in the mitochondria and it is an aerobic process requiring the
presence of oxygen. Increased fatty acid oxidation is a characteristic of starvation and
uncontrolled diabetes mellitus leading to ketone body formation by the liver
(ketosis).Because gluconeogenesis is dependent on fatty acid oxidation, any impairment of
fatty acid oxidation leads to hypoglycaemia.
Fatty acids are activated before being catabolised. Lehninger (2008:647) Harper (2003:180)
This is the only step in the complete degradation of fatty acids that requires energy from ATP.
In the presence of ATP and CoA, the enzyme acyl-CoA synthetase (thiokinase) catalyzes the
conversion of a fatty acid to an ‘’active fatty acid’’ or acyl-CoA. Long chain fatty acids do not
cross the inner membrane as acyl-CoA but as carnitine derivatives. Short and medium chain
fatty acids enter the mitochondria passively and get activated inside the mitochondria. Entry
of activated fatty acids in the mitochondria is the rate limiting step in fatty acid oxidation.
In β-oxidation, two carbons at a time are cleaved from acyl-CoA molecules, starting at the
carboxyl end. The cyclic reaction sequence generates FADH2 and NADH. The chain is
broken between α(2) and β(3)-carbon atoms – hence the name β-oxidation (Harper 2003:180).

R CH 2 CH 2 C O- Fatty acid
CoA-SH ATP

1 Acyl-CoA AMP + PPi

Synthetase
O
Acyl-CoA
R CH 2 CH 2 C ~S-CoA

Inner mit. membrane Carn. transporter

Fig. 1.1a Fatty acid oxidation reactions.

Continued next page.

9
 O
Acyl-CoA
R CH 2 CH 2 C ~S-CoA

2 Acyl-CoA dehydrogenase
FAD
2~ATP
O
Δ2-trans-Enoyl-CoA FADH2
CH CH C ~S-CoA
H2O
R
H2O Respiratory chain

Δ2-Enoyl-CoA
Hydratase OH O

3 R CH CH2 C ~S-CoA L(+)-3-Hydroxy-acyl-CoA

4
NAD+
L(+)-3-Hydroxyacyl-CoA 3~ATP
3-Ketoacyl-CoA O O Dehydrogenase
R C CH2 C ~S-CoA NADH + H+ Respiratory chain H2O
5
Thiolase O O

CH3 C ~S-CoA
R C ~S-CoA + Acetlyl-CoA
CoA-SH Acyl-CoA

CAC
2CO2

2 Acyl-CoA dehydrogenase

3 Δ2-Enoyl- CoA
hydratase

4 L-3-hydroxyacyl-CoA
dehydrogenase
5 Thiolase Fig. 1.1b Fatty acid
oxidation reactions

Long-chain acyl-CoA is cycled between reactions 2 to 5, acetyl-CoA being split off each
cycle by thiolase (Fig. 1.1a and Fig. 1.1b). When acyl radical is 4 carbon atoms in length, two
acetyl-CoA molecules are formed in the reaction 5. Oxidation of fatty acids produces a large
quantity of ATP. Eg In oxidation of palmitate- a 16 carbon fatty acid, FADH2 and NADH

10
will result in 5 high energy phosphates in the respiratory chain for each of the first 7 acetyl-
CoA molecules formed by β-oxidation (7 X 5 =35)
A total of 8 molecules of acetyl-CoA are formed and each will give 12 molecules of AT P on
oxidation in the citric acid cycle. Total (8 X 12= 96)
Two is subtracted from the initial activation of fatty acid yielding a net gain of 129 molecules
of ATP per mole of palmitate.

Ketone bodies are formed from acetyl-CoA when fat breakdown dominates. Acetyl-CoA
formed from fatty oxidation enters the citric acid cycle only if fat and carbohydrate
degradation are approximately balanced. The entry of acetyl-CoA into the citric acid cycle
depends on the availability of oxaloacetate for the formation of citrate - but concentration of
oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized-oxaloacetate is
normally formed from pyruvate, the product of glycolysis. In fasting or uncontrolled diabetes
mellitus, oxaloacetate is consumed to form glucose by the gluconeogenic pathway and hence
unavailable for condensation with acetyl-CoA. Under these conditions acetyl-CoA is diverted
to the formation of acetoacetate, D-3-hydroxybutyrate and acetone, the ketone bodies. The
divertion to formation of ketone bodies allows β-oxidation to continue because it frees CoA.
Abnormally high levels of ketone bodies are present in the blood of untreated diabetics.

The overall production of ATP per gram of palmitate is about twice that per gram of glucose,
because glucose is already partially oxidized compared to palmitate (Table 1.1). The caloric
value of fats is about twice that of sugars.

Substrate Mol. Wt Net ATP yield ATP Cal value

(mol/mol) (mol/g) (kJ/g
Glucose 180 36-38 0.2 17 Table 1.1

Palmitate 256 129 0.5 37

Ketone bodies serve as fuel for extrahepatic tissues. In extrahepatic tissues acetoacetate, is
activated to acetoacetyl-CoA by transferase (NB liver lacks this enzyme). CoA is transferred
from succinyl-CoA to form acetoacetyl-CoA. The acetoacetyl-CoA is split to acetyl-CoA by
thiolase and the formed acetyl-CoA is oxidized in the citric acid cycle (Fig. 1.2). While
acetoacetate and D-3-hydroxybutyrate are readily oxidized in the extrahepatic tissues, acetone
is difficult to oxidize in vivo and is to a large extent volatilized in the lungs. Heart muscle and
renal cortex tend to use acetoacetate for energy in preference to glucose. During prolonged
starvation and in diabetes the brain adapts to utilization of acetoacetate for energy supply.

11
 MUSCLE
FFA

Acetyl-CoA
LIVER
Acyl-CoA Thiolase
Acetoacetyl-CoA

Acetyl-CoA
Succinate OAA
Fig 1.2 Oxidation of
CoA
HMG-CoA transferase
succinyl-CoA
Citrate ketone bodies by
CO2 peripheral tissues
Acetoacetate
Acetoacetate
NADH + H+ NADH + H+
NAD+ NAD+
3-hydroxybutyrate
3-hydroxybutyrate
Transport of ketone bodies from liver and pathways of utilization and oxidation in
extrahepatic tissues

Biosynthesis of fatty acids

The main pathway for de novo synthesis of fatty acids occurs in the cytosol. The system is
present in many tissues including liver, kidney, brain, lung, mammary gland, and adipose
tissue. Acetyl –CoA is the precursor and free palmitate is the end product. In eukaryotes,
nearly all the acetyl-CoA used in fatty acid synthesis is formed in mitochondria from pyruvate
oxidation and from the catabolism of the carbon skeletons of amino acids. Acetyl –CoA from
oxidation of fatty acids does not represent a significant source of acetyl-CoA for fatty acid
biosynthesis in animals because the two pathways are reciprocally controlled. The
mitochondrial inner membrane is impermeable to acetyl-CoA, so an indirect shuttle transfers
acetyl group equivalents across the inner membrane. Citrate formed after condensation of
acetyl-CoA and oxaloacetate in the citric acid cycle is translocated into the cytosol via the
tricarboxylate transporter. In the presence of ATP and CoA , citrate is cleaved by ATP-citrate
lyase to acetyl- CoA and oxaloacetate. Released oxaloacetate forms malate in a reaction
catalyzed by NADH-linked malate dehydrogenase. The malate formed is converted to
pyruvate by malic enzyme with generation of NADPH a source of reductive power for fatty
acid synthesis (Fig 1.3).
Production of malonyl-CoA is the initial and controlling step in fatty acid synthesis.
Bicarbonate is a source of CO2 in the carboxylation of acetyl-CoA to malonyl-CoA in the
presence of ATP and enzyme acetyl-CoA carboxylase. Acetyl-CoA carboxylase is a
multienzyme complex which requires biotin for activity.

12
 Acetyl-CoA is the principal building block of fatty acids
Fatty acid synthesis

Pyruvate Glucose
Fatty acids
Mitochondrial
membrane
Pyruvate Fig 1.3 Acetyl-CoA as
dehydrogenase
Acetyl-CoA precursor for fatty acid
Acetyl-CoA
Oxaloacetate
synthesis.
ATP-citrate lyase NADH
Malate
Oxaloacetate dehydrogenase
Citrate
Citrate NAD+
Malate
CAC NADP+
Malic enzyme
Pyruvate NADPH*
carboxylase Pyruvate Pyruvate
CO2 CO2

The fatty acid synthase complex catalyzes formation of fatty acids. It is a dimer and each
polypeptide contains seven enzyme activities. The seven enzyme activities are malonyl
transacylase, acetyl transacylase, ketoacyl synthase, thioesterase, ketoacyl reductase,
enoyl reductase, and hydratase (Fig. 1.4). Acetyl-CoA used as a primer forms carbon atoms
15 and 16 of the final product palmitate. Addition of all the other 2-carbon units is via
malonyl-CoA. The final product in synthesis of fatty acids with an even number of carbon
atoms palmitate must be activated to acyl-CoA to proceed in other metabolic pathways.

The long carbon chains of fatty acids are assembled in a repeating four-step sequence.
Saturated acyl groups produced by this set of reactions become the substrate in subsequent
condensation with an activated malonyl group. With each passage through the cycle the fatty
acyl chain is extended by two carbons. When the chain length reaches 16 carbons the product
palmitate (16:0) leaves the cycle. The methyl and the carboxyl carbon atoms of the priming
acetyl group become C-16 and C-15 respectively, of the palmitate; the rest of the carbon
atoms are derived from acetyl-CoA via malonyl-CoA. The reducing agent in the synthetic
sequence is NADPH. All the reactions in the synthetic process are catalyzed by a
multienzyme complex, fatty acid synthase. Reactions are shown below (Fig. 1.4). In brief, A
priming molecule of acetyl-CoA combines with cysteine –SH group of ketoacyl- synthase
catalyzed by acetyl transacylase. Malonyl-CoA combines with the adjacent –SH on the 4’-
phosphopantetheine on an acyl carrier protein of the other monomer catalyzed by malonyl
transacylase to form acetyl (acyl)– malonyl enzyme. The acetyl residue reacts with malonyl
on acyl carrier protein with liberation of CO2 to form 3-ketoacyl enzyme (acetoacetyl
enzyme). The next steps in the sequence of reactions involve reduction, dehydration and
reduction again to form to the corresponding saturated acyl derivative. The sequence is
repeated six more times until a saturated 16-carbon acyl (palmityl) has been assembled. The
saturated 16-carbon acyl (palmityl) is released from the enzyme complex by a seventh
enzyme thioesterase. The free palmitate must be activated to acyl-CoA to proceed in other
metabolic pathways. Other metabolic pathways include esterification into triglycerides for

13
storage purposes, chain elongation or desaturation and esterification to cholesteryl ester.
Elongation and unsaturation of fatty acids occurs in the endoplasmic reticulum.

You may find fatty acid synthesis pathway confusing at first but for you to understand the
process fully please refer to the prescribed and recommended reading where there are
elaborate diagrams and sketches. Propionyl-CoA acts as a primer for synthesis of long-chain
fatty acids having odd number of carbon atoms.

Acetyl –CoA
Carboxylase
Acetyl -CoA Malonyl-CoA C3
C2 CO2 Malonyl-CoA
transacylase
Acetyl
transacylase

ENZYME Acyl(acetyl)-malonyl enzyme

COMPLEX
3 Ketoacyl -
synthase
3-Ketoacyl enzyme(acetoacetyl enzyme)

NADPH + H+ 3 Ketoacyl
reductase

NADP+
D-3-Hydroxyacyl enzyme

From PPP, Fig 1.4 Reactions in

isocitrate Hydratase
dehydrogenase, fatty acid synthesis.
2,3-Unsaturated acyl enzyme
malic enzyme

NADPH + H+ Enol reductase

NADP+
Acyl enzyme
Thioesterase
After cycling through
steps 2-5 seven times
Palmitate

The main source of NADPH for fatty acid synthesis is the pentose phosphate pathway. The
oxidative reactions of the Pentose Phosphate Pathway (PPP) are the chief source of hydrogen
required for reductive synthesis of fatty acids. Tissues specializing in active fatty acid
synthesis ie liver, adipose tissue, and lactating mammary gland also possess an active PPP
which also occurs in the cytosol and so there are no barriers against the transfer of NADPH.
Other sources of NADPH include the reaction that converts malate to pyruvate catalyzed
by NADP malate dehydrogenase ‘’ Malic enzyme’’ and the reaction catalyzed by
extramitochondrial isocitrate dehydrogenase (Harper 2003:176).

The nutritional state regulates fatty acid synthesis. Excess of carbohydrate is stored as fat in
many animals in anticipation of periods of caloric deficiency. Fatty acid synthesis converts
surplus glucose and intermediates such as pyruvate, lactate and acetyl-CoA to fat. It’s
depressed under low restricted caloric intake, or when there is deficiency of insulin – the later

14
conditions are associated with increased concentrations of free fatty acids in the plasma. The
nutritional state is the main factor regulating the rate of fatty acid synthesis. Fatty acid
synthesis is increased when sucrose is fed instead of glucose because fructose bypasses the
phosphofructokinase- 1 control point in glycolysis and floods the lipogenic pathway.

Short and long term mechanisms regulate fatty acid synthesis

Long-chain fatty acid synthesis is controlled in the short term by allosteric and covalent
modification of enzymes and in the long term by changes in gene expression governing rates
of enzyme synthesis. Acetyl-CoA carboxylase is the most important enzyme in the regulation
of fatty acid synthesis. This enzyme is activated by citrate and insulin and inhibited by
glucagon and epinephrine induced phosphorylation, long-chain acyl-CoA molecules (negative
feedback inhibition). Insulin also regulates lipolysis by other mechanisms. Increases
transport of glucose into the cell (adipose tissue), increasing the availability of pyruvate for
fatty acid synthesis and glycerol 3-phosphate for esterification.

1.7 Attempt the following questions below.

a) List the main steps, in a logical sequence of lipid digestion and absorption
b) What is the role of bile salts in lipid digestion and absorption?
c) Outline the steps in β– oxidation
d) Where does β– oxidation take place in a cell?
e) Which is the regulatory step in β– oxidation?
f) Name the ketone bodies.
g) In which part of the cell does fatty acid synthesis take place?
h) Where does NADPH for synthesis of fatty acids come from?
i) Where does elongation of fatty acids take place?
j) How is fatty acid synthesis regulated?

15
 UNIT 2

CHOLESTEROL METABOLISM

2.1 Introduction
There is a strong correlation between high levels of cholesterol in the blood and the incidence
of human cardiovascular diseases. It is also a major constituent of gall stones. Apart from
pathological complications it brings about, cholesterol is very useful For instance it forms an
important part of cell membranes where it contributes to the fluidity, a physiological
requirement for functional integrity of the membranes. Cholesterol is also a precursor of
steroid hormones and bile acids. The typical daily Western diet contains approximately 500
mg (1.2 mmol) of cholesterol daily, mainly in meat, eggs, and dairy products. Total plasma
cholesterol is about 5.2 mmol/L. Under normal circumstances, 30-60% of this is absorbed
from the gut (About half the cholesterol arises by biosynthesis and the remainder from diet).
Human beings synthesize 1g cholesterol each day, mainly in the liver. The rate of its
endogenous synthesis is determined by dietary intake. For this reason both dietary intake and
biosynthesis are important in determining its plasma concentration. Cholesterol is not required
in mammalian diet because all cells can synthesize it from simple precursors. About half the
cholesterol in the body is synthesized in the body and the remainder from diet. All tissues
with nucleated cells are capable of cholesterol synthesis, which occurs in the endoplasmic
reticulum and cytosol. Main dietary sources of cholesterol are foods of animal as mentioned
above.

2.2 Aim
The aim of the unit is to introduce to you the metabolism of cholesterol and its relationship to
disease processes

2.3 Objectives
By the end of the unit you should be able to:
1. Explain how dietary and endogenously synthesized cholesterol determine blood
cholestero.l
2. Explain how cholesterol synthesis is regulated.
3. Describe the relationship between LDL and HDL , and atherosclerosi.s
4. Explain how bile salts are formed.

2.4 Equipment
None

2.5 Other resources

None other than prescribed and recommended books

16
2.6 Contents

Biosynthesis of cholesterol

There are four stages of cholesterol synthesis

Stage 1 three molecules of acetyl-CoA are condensed to form a molecule of mevalonate (Fig.
2.1)

Cholesterol structure

Fig. 2.1

The first two reactions catalyzed by thiolase and 3-hydroxy-3-methylglutaryl CoA (HMG)-
CoA synthase respectively are reversible and do not commit the cell to the synthesis of
cholesterol. The cytosolic HMG-CoA synthase is dictinct from the mitochondrial isoenzyme
that catalyzes HMG-CoA synthesis in ketone body formation. The third reaction is the
committed step: the reduction of HMG-CoA to mevalonate catalyzed by HMG-CoA
reductase. It is a major point of regulation of cholesterol synthesis and is is the site of action
of most effective class of cholesterol –lowering drugs the HMG-CoA reductase inhibitors
(statins).Two molecules of NADPH donate two electrons each.

17
Stage 2 involves conversion of mevalonate into activated isoprene units.
Three phosphate groups are transferred from three ATP molecules to mevalonate. This leads
to formation of activated isoprene units ; Δ3-isopentenylpyrophosphate and dimethylally
pyrophosphate.

Stage 3 involves the polymerization of six 5-carbon isoprene units to form a 30 carbon linear
structure of squalene. Isopentenyl pyrophosphate and dimethylally pyrophosphate undergo
condensation, forming a 10 carbon chain , geranyl pyrophosphate. Geranyl pyrophosphate
undergoes undergoes another condensation with isopentenyl pyrophosphate yielding the 15-
carbon intermediate farnesyl pyrophosphate. Finally two molecules of farnesyl pyrophosphate
join forming squalene.

Finally , in stage 4 the cyclizationof squalene forms the four rings of the steroid nucleus, and
a further series of changes (oxidations, removal or migration of methyl groups) leads to the
final product cholesterol (Harper 2003:219).

Regulation of cholesterol biosynthesis.

Glucagon promotes phosphorylation

Acetyl-CoA and inactivation of HMG-CoA
reductase.

Insulin promotes dephosphorylation,

activating the enzyme favoring
3-hydroxy-3- cholesterol synthesis
methylglutaryl-CoA
+ Insulin
HMG-CoA reductase
Rate limiting step Glucagon
−

Mevalonate
Fig. 2.2

Cholesterol (intracellular)

Cholesterol synthesis is regulated by insulin and glucagon. Insulin promotes

dephosphorylation of the enzyme HMG-CoA reductase leading to its activation and
cholesterol synthesis. Glucagon on the other hand decreases cholesterol synthesis by
inhibiting HMG-CoA reductase through phosphorylation (Fig. 2.2).

When the total amount of cholesterol synthesized and obtained in the diet exceeds the
amounts required for synthesis of membranes, bile salts, and steroids, pathological
accumulation of cholesterol occurs leading to atherosclerosis. Atherosclerosis is linked to
high levels of low density lipoproteins (LDL)-bound cholesterol. There is also a negative

18
correlation between high density lipoproteins (HDLs) levels and arterial disease. HDLs act as
scavengers of cholesterol. In familial hypercholesterolemia blood levels of cholesterol are
very high and afflicted individuals develop severe atherosclerosis in childhood. LDL receptor
is defective in these individuals and the receptor-mediated uptake of cholesterol carried by
LDL does not occur. Therefore cholesterol is not cleared from the blood. Two products
derived from fungi (lovastatin and compactin) are used to treat patients with familial
hypercholesterolemia. The two compounds and several synthetic analogues are competitive
inhibitors of HMG-CoA reductase.

The fate of cholesterol

Much of cholesterol synthesis takes place in the liver. A small fraction is incorporated into the
membranes of hepatocytes. Most exported in one of the three forms:
 Biliary cholesterol
 Bile acids and salts - hydrophilic cholesterol derivatives that aid in lipid digestion
 Cholesterol esters – formed in the liver cholesterol and cholesterol esters are carried to
extrahepatic tissues by low-density lipoproteins (LDL).

Bile acids and salts

Synthesis of bile acids is one of the important mechanisms for the excretion of excess
cholesterol. An activating enzyme converts 7-hydroxycholesterol to bile acids which exist
within liver cells as colyl-CoA and chenodeoxycholyl-CoA derivatives. Another enzyme
catalyzes conjugation of activated CoA derivatives with glycine or taurine to form glycocolic
acid or glycochenodeoxycholic acid and taurocholic acid or taurochenodeoxycholic acid –
these are the primary bile acids (Fig. 2.3). In humans ratio of glycine to taurine conjugates is
3:1. At physiological pH the bile acids are mainly ionized and so occur as Na+ or K+ salts –
the terms bile acids and bile salts are sometimes used interchangeably.

19
Fig. 2.3 Synthesis of the 2 primary bile acids, cholic
acid and chenodeoxycholic acid. The reaction catalyzed
by the 7α-hydroxylase is the rate limiting step in bile
acid synthesis. Conversion of 7α-hydroxycholesterol to
the bile acids requires several steps not shown in detail
in this image. Only the relevant co-factors needed for
the synthesis steps are shown.
Deoxycholic and lithocholic bile acids are formed within the intestine through action of
bacterial enzymes. These enzymes hydrolyze the amide linkage of taurine and glycine. Up to
30g of bile acids per day enter the small intestine and only about 2% of these are lost in feces.
There is passive reabsorption of bile acids in the ileum and colon and active reabsorption in
the ileum
Bile acids are involved in four primary significant functions:
1. Their synthesis and subsequent excretion in the feces represent the only significant
mechanism for the elimination of excess cholesterol.
2. Bile acids and phospholipids solubilize cholesterol in the bile, thereby preventing the
precipitation of cholesterol in the gallbladder.
3. They facilitate the digestion of dietary triacylglycerols by acting as emulsifying agents
that render fats accessible to pancreatic lipases.
4. They facilitate the intestinal absorption of fat-soluble vitamins.

2.7 Attempt the following questions

a) Which important molecules are derived from cholesterol?
b) Explain how cholesterol synthesis is regulted by insulin and glucagon.
c) Why is dietary restriction alone not effective in control of blood cholesterol?
d) Explain how endogenous synthesis of cholesterol is regulated
e) What role do bile salts play in digestion?

20
 UNIT 3
AMINO ACID METABOLISM

3.1 Introduction
What is amazing about proteins is the fact that even though they are in billions in human
body, they are all made from the same twenty amino acids. These 20 amino acids are able to
form many proteins because of the variations in their side chains, the order in which they
appear in a polypeptide and the length of the polypeptides. As discussed in module one, the
side chains of amino acids determine their behavior which in turn determines the type and
behavior of proteins they form. In addition to their roles as building blocks for peptides and
proteins and as precursors of neurotransmitters and hormones, amino acids are also a source
of energy from diet and during fasting. This is because the carbon skeletons of some amino
acids can be oxidized or used to form glucose through gluconeogenesis, providing a metabolic
fuel for tissues that prefer glucose as a source of energy. In individuals consuming adequate
amounts of protein a significant quantity of amino acids may be converted to carbohydrate
(glycogen) or fat (triglycerides) for storage. Unlike carbohydrates amino acids do not have a
dedicated storage form equivalent to glycogen. When amino acids are metabolized the amino
group is excreted as urea and the carbon skeleton is used in other metabolic processes.

3.2 Aim
The aim of the unit is to introduce you to metabolism of amino acids.

3.3 Objectives
By the end of the unit you should be able to:
1. Define essential and non essential amino acids.
2. List essential and non essential amino acids.
3. Describe catabolism of amino acid nitrogen.
4. Explain the central role of glutamate and glutamine to amino acid metabolism.
5. Outline the reactions of the urea cycle.

3.4 Equipment
None

3.5 Other resources

a) [Link]
b) [Link]
c) [Link]
d) [Link]

3.6 Contents

Amino acid synthesis.

As pointed out earlier there are 20 amino acids found in human protein. Out of these twenty
amino acids, humans can synthesize 10 of them. These 10 which are synthesized by humans

21
do not need to be supplied in diet. To put it in a different way, they are dietary nonessential;
hence the name nonessential amino acids. Of the 10 nonessential amino acids, 7 are formed
from amphibolic intermediates and 3 (cysteine, tyrosine and hydroxylysine from) from
nutritionally essential amino acids.
Essential amino acids are those amino acids that can not be synthesized in human body and
must be supplied in the diet. These amino acids are dietary essentials; hence the name
essential amino acids. The table below shows the essential and nonessential aminoacids.

Essential Nonessential
Arginine* Alanine
Histidine Asparagine
Isoleucine Aspartate
Leucine Cysteine
Lysine Glutamate
Methionine Glutamine
Phenylalanine Glycine
Threonine Hydroxyproline Not necessarily for protein synthesis-formed
Tryptophan Hydroxylysine during processing of collagen
Valine Proline
Serine
Tyrosine
* Histidine is categorized as nutritionally semiessential because in infants it is inadequately
synthesized to support growth.

Nutritionally nonessential amino acids have short biosynthetic pathways.

The carbon skeletons of all twenty amino acids are derived from just seven metabolic
intermediates, which are found in three metabolic pathways. These metabolic pathways are:
1) Glycolysis provides three intermediates for amino acid synthesis and these are 3-
phosphoglycerate, phosphoenolypyruvate, and pyruvate,
2) The pentose phosphate pathway provides two intermediates for amino acid synthesis
and these are ribose 5-phosphate and erythrose 4-phosphate
3) The citric acid cycle provides two intermediates and these are -ketoglutarate and
oxaloacetate.
In general, the structures of the essential amino acids are more complex than the nonessential
amino acids which is reflected in the number of enzymatic reactions required for synthesis.
Formation of nonessential amino acids involves short biosynthetic pathways as shown below
(1 to 8)
1. Glutamate and glutamine
α-ketoglutarate L-Glutamate
Glutamate dehydrogenase
L-Glutamate L-Glutamine
Glutamine synthetase

2. Serine –is formed from glycolytic intermediate 3-phosphoglycerate

3. Glycine – Glycine aminotransferases catalyze synthesis of glycine from glyoxylate
and glutamate or alanine

22
 4. Proline – Formed from glutamate
5. Cysteine – Formed from essential methionine
6. Tyrosine – Formed from essential phenylalanine
7. Alanine – Formed from transamination of pyruvate
8. Aspartate and asparagine
Aspartate is formed from transamination of oxaloacetate. Asparagine is formed from
aspartate in a reaction catalyzed by asparagine synthetase

Amino acids are also a source of intermediates and some biologically essential molecules
other than proteins. For instance pyruvate can be formed from glycine, serine, alanine,
cysteine, threonine, 4-hydroxyproline. Acetyl CoA can be formed from tyrosine,
phenylalanine, lysine and tryptophan. Enzymatic deficiencies in metabolism of these amino
acids to acetyl-CoA can lead to metabolic diseases. Alkaptonuria is a metabolic diseases of
tyrosine metabolism and its caused by deficiency of homogentisate oxidase. In this condition
urine darkens on exposure to air. Phenyalanine is first converted to tyrosine and subsequent
reactions are those of tyrosine. Phenylketonuria is a metabolic disorder of phenylalanine
metabolism due to deficiency of phenylalanine hydroxylase. Hartnup disease is a metabolic
disorder of tryptophan metabolism characterized by pellagra like skin rash and mental
retardation. Other examples of amino acids being converted to metabolic intermediates are
cited under the topics citric acid cycle and gluconeogenesis covered in the previous module.

Metabolic disorders of branched amino acids(Leucine, valine and isoleucine)

The branched amino acids are not metabolized in the liver. They are metabolized in
extrahepatic tissues with an aminotransferase not found in the liver that acts on all three
branched-chain amino acids to produce corresponding α-ketoacids. The branched-chain α-
keto acid dehydrogenase complex then catalyzes the oxidative decarboxylation of all three
α-ketoacids, in each case releasing a carboxyl group as CO2 and producing the acyl-CoA
derivative (Analogous to reactions catalyzed by pyruvate dehydrogenase and α–ketoglutarate
dehydrogenase). The three enzyme systems have the same cofactors (NAD, FAD, TPP,
lipoate, CoA).In maple syrup urine disease (because of the characteristic odor imparted to
the urine by the α-ketoacids ) the three branched-chain α-ketoacids accumulate in the blood
and spill over in the urine. This condition results from defective branched-chain α-keto acid
dehydrogenase complex. Untreated this condition leads to abnormal development of the
brain, mental retardation, and death in early infancy. Treatment involves limiting the amounts
of valine leucine and isoleucine to the minimum required for normal growth.

Some molecules other than proteins derived from amino acids include porphyrins where
glycine is a precursor, phosphocreatine (synthesis of phosphocreatine requires glycine,
arginine and methionine), glutathione (synthesis of glutathione requires glutamate, cysteine
and glycine.). Amino acids are also converted to biologically useful amines for instance
tyrosine forms epinephrine, glutamate forms γ,–aminobutyrate, histidine forms histamine, and
tryptophan forms serotonin.

23
Catabolism of amino acid nitrogen
Excess amino acids can not be stored nor are they excreted as such. The amino acids are
metabolized such that the amino groups are removed by transamination or oxidative
deamination and the remaining carbon skeletons are used as intermediates for other reactions.
Fish are ammonotelic because they excrete amino groups as ammonia. Birds and amphibians
are uricoteric because they excrete amino groups as uric acid and mammals are ureotelic
because they excrete urea. Ammonia is toxic to the nervous system and uric acid results in
crystal deposition. So to avoid theses effects humans evolved a capacity to convert their
nitrogenous waste to the highly soluble nontoxic urea (Harper 2003:242).

Urea biosynthesis occurs in four stages. These are:

1. transamination
2. oxidative deamination of glutamate
3. ammonia transport
4. reactions of the urea cycle

Transamination
Transamination catalyzed by aminotransferases interconverts a pair of amino acids and a pair
of keto acids. Coenzyme for aminotransferases is pyridoxal phosphate (PLP). Through
transamination amino acids can transfer there amino groups to keto acids like pyruvate and α–
ketoglutarate to form alanine and glutamate respectively (Fig. 3.1). Alanine is a carrier of
these amino groups from muscles to the liver. In the liver α–ketoglutarate is the acceptor of
most of the amino groups from other amino acids to form glutamate. Glutamate then enters
the mitochondria where it undergoes oxidative deamination to release ammonia and reform α–
ketoglutarate in the process

Fig. 3.1 Transaminations are catalyzed by transaminases which interconvert a pair of

amino acids and a pair of ketoacids

Oxidative deamination
As pointed out above amino groups of most amino acids ultimately are transferred to α-
ketoglutarate by transamination forming glutamate in the cytosol. Glutamate then enters the
mitochoindria where nitrogen is released as ammonia through oxidatice deamination(Fig.
3.2). Release of this nitrogen as ammonia is catalyzed by glutamate dehydrogenase. Amino
acid oxidases also remove nitrogen as ammonia. Glutamine which carries amino groups from

24
peripheral tissues is also deaminated in the mitochondria to form glutamate which is then
metabolized as explained above.

Metabolic fates of amino groups

Cellular protein
LIVER
Amino
acids COO− COO−
from +
ingested H3N C H C O
protein
R R α-keto acids
Amino acids

COO− COO−
+
C O H3N C H
CH2 CH2 Glutamate
α-ketoglutarate
CH2 CH2
COO− COO−
Alanine COO−
from + Gluta
muscle
H3N C H
COO− mine
+ COO− NH4+ CH2 from
H3N C H muscle
C O CH2 and
CH3 Glutamine
CH3 other
Alanine C
Pyruvate Ammonia, NH2 tissues
O 3
urea , uric acid .

Fig. 3.2 Metabolic fates of amino groups

Formation of ammonia and its toxicity

Ammonia produced by enteric bacteria and absorbed into the portal venous blood and the
ammonia produced by tissues are rapidly removed from the circulation by the liver and
converted to urea. Should portal blood bypass the liver systemic blood ammonia may rise to
toxic levels. This occurs in severe hepatic impairment like cirrhosis where collateral
circulation is [Link] of ammonia toxicity include tremor, slurred speech,
blurred vision, coma, and ultimately death.
Urea synthesis
The ammonia formed upon deamination of glutamate and glutamine is converted to the non-
toxic form urea. The major pathway of nitrogen excretion in humans is urea synthesized by
the liver, released into the blood, and cleared by the kidneys. Of the six amino acids involved
in urea synthesis, N-acetyl glutamate functions as an enzyme activator, remaining 5-
aspartate, arginine, ornithine, citrulline and argininosuccinate function as carriers of atoms.
Amino acids aspartate and arginine occur in proteins but ornithine, citrulline and
argininosuccinate do not occur in proteins but play a role in urea synthesis (Fig. 3.3).

25
 From glutamine and
glutamate

Urea
CO2 + NH4 Argininase
Carbamoyl 5
phosphate 1
synthase 1 Ornithine

Carbamoyl Ornithine Arginine

phosphate transcarba
moylase 4
2 Fumarate
Argininosuccinase

Citrulline Argininosuccinate
Argininosuccinic
acid synthase
Rate
limiting Fig. 3.3 The urea cycle
3
enzyme
Aspartate

The activity of the urea cycle is regulated at two levels

• The flux of nitrogen through the urea cycle varies with the organism's diet
– On high protein diet, the carbon skeletons of amino acids are used for fuel and
excess amino groups are converted into urea.
– During prolonged starvation, breakdown of muscle protein supplies much of
the metabolic energy and this results in increased urea production.
– All five enzymes involved in urea production are synthesized at higher rates in
starving animals and in animals on high-protein diet
• Allosteric regulation of carbamoyl phosphate synthetase I. This enzyme is activated
by N-acetylglutamate

3.7 Attempt the following questions below

a) Which amino acids can form pyruvate?
b) Which enzyme is defective in phenylketonuria?
c) Which amino acid undergoes oxidative deamination in the mitochondria more than any
other ?

26
d) Under what conditions is urea synthesis increased?
e) Which amino acids do not occur in human protein but participate in urea synthesis?
f) Outline the reactions of the urea cycle.

27
 UNIT 4
NUCLEIC ACID METABOLISM

4.1 Introduction
For the last several weeks you have learnt the roles of proteins in cells. You have seen that
their function depends entirely on their structure. Where does that structure come from? In the
next couple of topics you will see how information relating to the structure of proteins is
stored as a sequence of nucleotides in DNA. You will also see how a system decodes this
information and reliably produces protein molecules with certain structures and functions.
Knowledge of these processes and their meaning form the basis of inheritance and
[Link] structures of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were
covered in general biochemistry and therefore will not be discussed here in detail. Cellular
nucleic acids exist in two forms, DNA and RNA. Approximately 90% of nucleic acids within
cells is RNA, and the remainder DNA. For cells to divide DNA must be duplicated. The
process starts with one double-stranded DNA molecule and produces two identical copies of
the molecule. The replication of DNA is a necessary preliminary step for cell division. It
occurs during interphase of the cell cycle, in which a second chromatid containing a copy of
the DNA molecule is assembled. Each strand of the original double-stranded DNA molecule
serves as template for the production of the complementary strand. This method of DNA
replication is described as ´semi-conservative´. Simply stated, transcription is the synthesis of
RNA from a DNA template. For you to understand the topic fully your will need to refer to
the references and other resources given where there are more elaborate diagrams.

4.2 Aim
The aim of the unit is to introduce the basic concepts of nucleic acid metabolism.

4.3 Objectives
By the end of the unit you should be able to:
1. List the steps involved in DNA replication.
2. Explain the role played by each of the enzymes involved in DNA replication.
3. List the main types of RNA.
4. Describe the similarities and differences between DNA and RNA synthesis.

4.4 Equipment
None

4.5 Other resources

a) [Link]
b) [Link]
c) [Link]
d) [Link]

28
4.6 Contents

DNA synthesis
As pointed out earlier DNA is a double stranded nucleic acid with the two strands running in
antiparallel directions. DNA synthesis is semiconservative meaning that after replication the
resulting double strand has one parental and one newly synthesized or daughter strand.
There are six steps involved in DNA replication in eukaryotes
1. The binding of special proteins a the origin of replication of a DNA template
2. Unwinding (denaturation of double stranded DNA to provide a single strand DNA
template)
3. Formation of replication fork
4. Initiation of DNA synthesis and elongation
5. Formation of replication bubbles with ligation of newly synthesized DNA fragments
6. Reconstitution of the chromatin structure

Origin of replication
At the origin of replication there is an association of sequence specific binding proteins with a
series of repeat DNA sequences to form a complex.

Unwinding of DNA
Interaction of proteins with origin of replication provides a short region of single stranded
DNA essential for initiation of synthesis of nascent DNA strand. DNA helicase allows ATP-
driven processive unwinding of DNA. Single stranded DNA – binding proteins stabilize the
complex preventing reannealing of the single strands to double strands.

Formation of replication fork

Replication fork consists of four components (Fig. 4.1)
1. DNA helicase unwinds a short segment of the parental duplex DNA
2. A primase initiates synthesis of mRNA molecule that is essential in priming DNA
synthesis. The mRNA must be complementary with the DNA strand
3. The DNA polymerase initiates nascent, daughter strand synthesis
4. Single strand(ss) DNA-binding proteins bind to single strand DNA and prevent
premature reannealing of the ssDNA to double stranded DNA without affecting the
abilities of nucleobases to serve as templates (Harper 2003:328).
Polymerase III is the main enzyme that polymerizes nucleotides to the growing chain. It is a
very large enzyme that catalyzes synthesis of two daughter strands at the same time. This
enzyme binds to DNA template. It has high processivity which means it catalyzes formation
of many thousands of phosphodiester bonds before releasing its template. It also has got high
catalytic power which means it can add about 1000 nucleotides per minute. It’s a dimer
because both strands of parental DNA must be replicated at the same place and at the same
time. The enzyme is asymmetrical because both the leading and the lagging strands are
synthesized differently. DNA polymerases only synthesize DNA in the 5’ to 3’ direction.
Because the DNA strands are antiparallel, the polymerase functions asymmetrically. An
enzyme capable of polymerizing DNA in the 3’ to 5’ position does not exist which means
both newly synthesized strands can not grow in the same direction. To go round this problem

29
on the leading (forward) daughter strand the DNA is synthesized continuously in the 5’ to 3’
direction and on the lagging (retrograde) strand, the DNA is synthesized in short 1-5 kb
fragments known as the Okazaki fragments, in the opposite direction to the leading
(forward) strand, in the 5’ to 3’ direction, facing the back end of the preceding RNA primer.
Okazaki fragments are synthesized in sequence for each replication fork. To ensure this, the
helicase loops the template strand for the lagging strand. The lagging strand template would
then pass through the polymerase site in one subunit of dimeric poly III in the same direction
as the leading strand template in the other subunit. The DNA Pol III lets go of the lagging
strand after about 1000 nucleotides have been added. A new loop would then be formed and
tne process repeats itself. The helicase associates with the primase to afford the latter proper
access to the template and this allows the RNA primer to be made, and in turn the polymerase
to begin replicating DNA. The mobile complex between helicase and primase is called
primosome.

Helicase

Primosome
Single-strand Fig. 4.1 Proteins
binding protein involved in DNA
3’ Primase replication

DNA polymerase I

DNA
polymerase III 5’
3’ 3’
5’
Lagging strand

Leading strand
DNA ligase

As the synthesis of an Okazaki fragment is completed and the polymerase is released, a new
primer has been synthesized and the same polymerase molecule remains associated with the
replication fork and proceeds to synthesize the next Okazaki fragment. There are three main
types of DNA polymerase. DNA polymerase III catalyzes the highest rate of chain elongation
and is the most processive whereas DNA polymerase II is involved in proof reading and
repair. DNA polymerase I completes chain synthesis between Okazaki fragments on the
lagging strand and erases the primer.

30
 Classes of proteins involved in replication
Protein Function
DnaA Recognizes origin sequence; opens duplex at specific sites
in the origin
Helicases (DnaB) Processive unwinding of DNA.

DnaC Required for DnaB binding at the origin

Single-strand binding protein Prevents premature annealing of dsDNA

DNA primase Initiates synthesis of RNA primer

DNA polymerase Deoxyribonucleotide polymerization

Topoisomerases Relieve torsion strain that results from helicase-

induced unwinding
DNA polymerase I Erases the primer. Completes DNA synthesis
DNA ligase Seals the single strand nick between the nascent
chain and the Okazaki fragment on lagging
strand

Initiation and elongation

Initiation requires priming by a short length of mRNA (10-200nucleotides long)
This is followed by nucleophilic attack by the 3’-hydroxyl group of the RNA primer on
the α phosphate of the first entering deoxyribonucleoside triphosphate with splitting off of
pyrophosphate. The 3’-hydroxyl group of the recently attached deoxyribonucleoside
monophosphate is free to carry out a nucleophilic attack on the next entering
deoxyribonucleoside triphosphate at the α phosphate moiety with splitting off of
pyrophosphate (Fig. 4.2). The selection of deoxyribonucleoside triphosphate to be attached is
dependent upon proper base pairing. By this stepwise process the template dictates which
deoxyribonucleoside triphosphate is complementary and by hydrogen bonding holds it in
place while the 3’-hydroxyl group of the growing strand incorporates a new nucleotide into
the polymer . In mammals after many Okazaki fragments are generated, the replication
complex begins to remove the RNA primers (removed by the 5’ to 3’ exonuclease activity
of Polymerase I), to fill the gaps with proper base paired deoxynucleotides (Polymerase I) and
then to seal the fragments of newly synthesized DNA by DNA ligases (Harper 2003: 329).

31
 Assembly of informational macromolecules require energy

Polynucleotide O
– Precursors for DNA are
chain O P O
nucleoside triphosphate.
O Polymerization is
CH2 Base accompanied by cleavage of
O the phosphoanhydride bonds
H H
between α and β phosphates,
H .OH OH
H
with release of PPi. Moieties
transferred to the growing
–
O O O chain are adenylates for
– γ β α RNA and deoxy derivatives
O P O P O P O
– – for DNA.
O O O

CH2
Fig. 4.2
Base
O
H H
O O H H
OH OH
– –
O P O P O
– –
O O

2Pi

Formation of replication bubbles

If replication was to occur from a single origin in mammals, it would take about 150 hrs.
The entire human genome replicates in about 9 hours. This is achieved by the multiple origins
of DNA replication that occur in clusters of about 100 (poorly defined sites). Replication
occurs in both directions up and down the chromosome and on both strands simultaneously.
This replication process is what generates ‘replication bubbles’ (Fig. 4.3).

32
 Fig. 4.3

Reconstruction of chromatin
Chromatin structure is reformed after replication

RNA synthesis
General steps required to synthesize the primary transcript are initiation, elongation and
termination. When DNA is transcribed into RNA, three different kinds of RNA can be
identified i.e. mRNA, tRNA, and rRNA. If there are changes in the nucleotides in DNA as a
result of damage or mutations , this can be reflected in the RNA transcript and its translation
would result in abnormal protein product. RNA molecules synthesized in mammalian cells are
made as precursor molecules that have to be processed into mature, active RNA. Errors or
changes in synthesis, processing, and splicing of mRNA transcripts can cause disease.
There are four major classes of RNA
1. Ribosomal RNA (rRNA)
2. Messenger RNA (mRNA)
3. Transfer RNA (tRNA)
4. Small nuclear RNA (snRNA)
RNA is synthesized from a DNA template by an RNA polymerases. There are some
similarities between RNA and DNA synthesis

Some similarities between DNA and RNA synthesis

1. Steps of initiation, elongation and termination with 5’ to 3’ polarity.
2. Large multicomponent initiation complexes.
3. Adherence to base pairing rules.

33
Some differences
1. Ribonucleotides are used in RNA rather than deoxyribonucleotides.
2. Uracil replaces Thymine as the complementary base pair for Adenine in RNA.
3. A primer is not involved in RNA synthesis.
4. A small portion of the genome is transcribed or copied into RNA, where as the entire
genome must be copied during DNA replication.
5. No proof reading function during RNA transcription.

In transcription, template strand of DNA is copied as RNA. The sequence of ribonucleotides

in an RNA molecule is complementary to the sequence of deoxyribonucleotides in one strand
of the double-stranded DNA molecule. The strand that is transcribed into a RNA molecule is
the template strand and the other is the coding strand (with the exception of T for U changes it
corresponds exactly to the sequence of the primary transcript). The template strand for each
gene will not necessarily be the same strand of the DNA double helix. A given strand of the
double-stranded DNA molecule will serve as the template strand for some genes and the
coding strand for other genes. The nucleotide sequence of an RNA transcript will be the same
(except for the U replacing T) as the coding strand. The information in the template strand is
read out in the 3’ to 5’ direction. DNA – dependent RNA polymerase initiates transcription at
a distinct site, the promoter. Mammalian cells possess three distinct nuclear DNA-dependent
RNA polymerase as shown below.

Form Sensitivity to α –Amanitin Major products

I (A) Insensitive rRNA

II (B) High sensitivity mRNA

III (C) Intermediate sensitivity tRNA/5S rRNA

α –Amanitin from mushroom Amanita phalloides is a specific differential inhibitor of

eukaryotic nuclear DNA-dependent RNA polymerase. Through their sensitivity to amanitin,
the three kinds of RNA polymerase can be differentiated (Harper 2003:343).

Initiation
A specific protein factor ( the sigma [σ] factor) assists the RNA polymerase to attach more
tightly to the deoxyribonucleotide of the promoter region. First nucleotide (almost always a
purine) then associates with the initiation site on the enzyme. In the presence of the
appropriate nucleotide, the RNA polymerase catalyzes the formation of a phosphodiester
bond. The enzyme polymerizes the ribonucleotides in a specific sequence dictated by the
template strand and interpreted by the base-pairing rules.

Elongation
DNA unwinding occurs in order to provide access for appropriate base pairing to the
nucleotides of the template strand.

Termination
Termination of RNA synthesis is signalled by a sequence in the template strand of the DNA
molecule. A termination signal is recognized by a termination protein, the rho factor. The
core enzyme separates from the DNA template. With the assistance of another σ factor, the
core enzyme recognizes another promoter at which synthesis of new RNA molecules

34
commences. As pointed out in earlier, newly synthesized RNA strands undergo processing
within the nucleus before they are released in the cytosol as mature RNA.

Synthesis of DNA from RNA

This process occurs in retroviruses. It is the reverse of what has been described above in that
here, the RNA serves as a template for synthesis of DNA. RNA-dependent DNA polymerase
(reverse transcriptase) is capable of synthesizing a single-stranded and then a double-stranded
DNA molecule from a single stranded RNA template. First a DNA-RNA hybrid is
synthesized using the RNA as template. Rnase H degrades the RNA template strand. The
remaining DNA strand in turn serves as a template to form a double-stranded DNA molecule
containing the information originally present in the RNA genome of the virus.

4.7 Please attempt the questions below

a) What is meant by ´DNA synthesis is semiconservative´?
b) What is the role of DNA polymerase III in DNA replication?
c) How do replication bubbles affect DNA synthesis?
d) What are the major products of DNA – dependent RNA polymerase I, II and III?
e) What type of reaction does RNA-dependent DNA polymerase (reverse transcriptase)
catalyze?

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 UNIT 5
PROTEIN SYNTHESIS

5.1 Introduction

Protein synthesis is important because it is through the use of genetic information to

synthesize proteins that the genes in the DNA are expressed in the phenotype. Protein
synthesis involves two big steps and several other smaller steps depending on the organism.
The two big steps are called transcription which has already been covered in the previous unit
and translation. Transcription involves the synthesis of an RNA transcript using the cell's
DNA as a template, leading to messenger RNA(mRNA). In eukaryotic cells transcription
happens in the nucleus. Once the messenger RNA is made it then is transported to the
cytoplasm where the information contained in the mRNA is translated into a sequence of
amino acids making up a polypeptide. Nucleotides found in DNA are adenylate, guanylate,
thymidylate and cytidylate represented by the letters A, G, T and C respectively. These
nucleotides are organized in 3 letter codes called codons. A collection of codons makes the
genetic code. The genetic code can provide information about protein defects, diagnosis and
treatment, and how viruses disrupt protein synthesis. There are 64 possible triplet codons (i.e.
43); 61 of these code for amino acids. AUG, the methionine codon, is the START codon, or
the one that initiated the process of translation. UAG, UGA, and UAA are STOP codons. The
genetic code is universal (with minor differences in mitochondria and ciliated protozoa) and
degenerate (i.e. more than one codon coding for one amino acid). Genetic information flows
from DNA to RNA to protein. In other words information stored in DNA is transferred to
RNA and then expressed in the structure of proteins. Translation from mRNA requires an
adaptor molecule which must recognize a specific nucleotide sequence on mRNA and must
also recognize a specific amino acid on the other hand. Functional groups of the amino acids
do not come into contact with the mRNA template. This adaptor molecule is transfer RNA
(tRNA) (Harper 2003: 358).

5.2 Aim
The aim of the unit is to familiarize you with the processes that are involved in translation of
information in DNA into a polypeptide chain.

5.3 Objectives
By the end of the unit you should be able to:
1. Describe the genetic code.
2. List the stages in protein synthesis:
3. Outline the flow of information from DNA in the nucleus to newly synthesized
polypeptide chain:
4. Describe the importance of post transilational modification:

5.4 Equipment
None

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5.5 Other resources
[Link]
[Link]
[Link]
[Link]

5.6 Contents
Protein synthesis occurs on ribosomes and there are basically five stages involved in protein
synthesis.
Stage 1. Activation of amino acids. Two fundamental requirements must be met. The
carboxyl group of each amino acid must be activated to facilitate formation of a peptide bond.
The link must be established between each amino acid and the information that encodes it in
the mRNA. Both requirements are met by attaching the amino acid to tRNA in the first stage
of protein synthesis. This takes place in the cytosol, not on the ribosome. Each of the twenty
amino acids is covalently attached to a specific tRNA at the expense of an ATP using Mg 2+
dependent enzymes called aminoacyl-tRNA synthetases. When amino acids are attached to
their respective tRNA they are said to be ‘charged’(Fig. 5.1)

Fig. 5.1 Amino acid activation

Stage 2. Initiation. The mRNA bearing the code for the polypeptide binds to the smaller of the
two ribosomal subunits and to the initiating aminoacyl-tRNA. The larger ribosomal subunit
then binds to form an initiation complex. The initiating aminoacyl-tRNA base-pairs with the
mRNA codon AUG that signals the beginning of the polypeptide. This process which requires
GTP, and is promoted by cytosolic proteins called initiation factors.

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 Fig 5.2 Translation

Stage 3 Elongation. Nascent polypeptide is lengthened by covalent attachment of successive

amino acid units each carried to the ribosome and correctly positioned by its tRNA, which
base-pairs to its corresponding codon in the mRNA. Covalent attachments of amino acids is
effected by the enzyme peptidyltransferase. Elongation requires cytosolic proteins called
elongation factors. The binding of each incoming aminoacyl-tRNA and movement of
ribosomes along mRNA are facilitated by hydrolysis of GTP. In other words the anticodon of
next tRNA binds to mRNA codon at A site of ribosome (see illustration below). The order of
bases in mRNA codons determines which tRNA anticodons will align which in turn
determines order of amino acids in protein. Amino acid at A site is linked to previous amino
acid through peptide bond. Ribosome moves along one codon and next tRNA binds at A site
(Fig. 5.2).

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Stage 4. Termination and release. Completion of the polypeptide chain synthesis is signaled
by a termination codon in the mRNA. The new polypeptide is released from the ribosome
aided by release factors (see illustration below)

Stage 5. Folding and posttranslational processing. To achieve its biological function the new
polypeptide must be folded into its proper three-dimensional conformation. Before or after
folding the polypeptide may undergo enzymatic processing. This may include removal of one
or more aminoacids from the amino terminus, addition of acetyl, phosphoryl, methyl,
carboxyl or other groups to some amino acid residues, proteolytic cleavage, and attachment of
oligosaccharides or prosthetic groups.

Knowledge of protein synthesis has aided the development of several antimicrobial drugs
which act through various mechanisms. The differences in protein synthesis between
microorganisms and humans have made these agents selective in their antimicrobial
properties. See below examples of some commonly used drugs with antimicrobial properties.

Some antibiotics inhibit protein synthesis through various mechanisms:

a) Streptomycin and other aminoglycosides .Interfere with the binding of
formylmethionyl-tRNA to ribosomes and prevent correct initiation.. This causes
misreading of mRNA
b) Tetracycline binds to the 30S subunit and inhibits binding of aminoacyl-tRNA
c) Chloramphenicol inhibits the peptidyl transferase activity of the 50S ribosomal
subunit
d) Erythromycin binds to the 50S subunit and inhibits translocation
e) Puromycin causes premature chain termination by acting as an analogue of aminoacyl-
tRNA.

5.7 Please attempt the following questions.

a) Which RNA molecules carry genetic information from the nucleus to protein
synthesizing machinery?
b) What is a stop codon?
c) What are two fundamental requirements for amino acid activation?
d) Which enzyme forms peptide bonds during polypeptide or protein synthesis?
e) How do different antibiotic inhibitors affect protein synthesis?

Summary of the module

Unlike carbohydrate fuels, which enter the body primarily as glucose or sugars that can be
easily converted to glucose lipid fuels are heterogeneous with respect to chain length,
branching and unsaturation. The catabolism of fatty acids is primarily a mitochondrial
process. The primary pathway of catabolism, of fatty acids involve their oxidative cleavage in
two-carbon units, a process known as β-oxidation which produces acetyl-CoA. In muscle the
acetyl-CoA units are used for ATP production in the mitochondria, where as in liver the
acetyl-CoA is catabolized to ketone bodies that are exported for energy production in
peripheral tissues. Fatty acid synthesis and storage are essential components of body energy
homeostasis. The process takes place in the cytosol and the main step is catalyzed by acetyl-
CoA carboxylase. The primary product of fatty acid synthesis is a 16 carbon saturated

39
palmitate which undergoes modification to form different fatty acids. These modifications
may include chain elongation and unsaturation.
Cholesterol is a vital constituent of cell membranes and the precursor molecule for bile acids,
steroid hormones, and vitamin D. Cholesterol is derived from the diet and also synthesized de
novo from acetyl-CoA. The rate limiting enzyme is HMG-CoA reductase. The metabolism of
cholesterol into bile acids and steroid hormones involves several hydroxylation reactions
catalyzed by cytochrome P450 monooxygenase enzymes. Several clinical disorders are
associated with abnormalities in the regulation of cholesterol homeostasis and metabolism.

The metabolism of amino acids is intimately associated with main stream metabolism. The
catabolism of amino acids generally begins with removal of the amino group which is
ultimately excreted as urea. The resulting carbon skeletons after removal of amino groups are
converted to intermediates that enter central metabolism at various points. Because carbon
skeletons corresponding to various amino acids can be derived from or fed into the glycolytic
pathway, citric acid cycle, fatty acid biosynthesis and gluconeogenesis, amino acid
metabolism should not be considered as an isolated pathway. Although amino acids are not
stored like glucose (glycogen) or fatty acids(triglyceride), they have an important and
dynamic role not only in providing the building blocks for synthesis of proteins but also in
normal energy metabolism, providing a carbon source for gluconeogenesis.

In DNA synthesis genetic information is replicated in a semi-conservative manner in which

parental strands are separated and both serve as templates for daughter strands. The major
products of transcription are rRNA, tRNA, mRNA which have specific functions in the cell.
Protein synthesis is the culmination of the transfer of information from DNA to proteins. This
information is carried from the nucleus to protein synthesizing machinery by mRNA. When
this information is translated on ribosomes a polypeptide chain is formed. This requires
accurate interaction between mRNA, tRNA and respective amino acids so that the right amino
acids can be covalently attached to the correct growing chain. Mutations in DNA result in
defective mRNA transcript which on translation can form abnormal protein with impaired
function resulting in disease.

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