Biochemistry Concept Book Atf

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com
Concept Based Learning
Video Companion on Each Chapter
Next Generation
Comprehensive Review Series

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“CRS BIOCHEMISTRY”
Active Recall Based

Integrated Edition
Published by Delhi Academy of Medical Sciences (P) Ltd.
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ISBN :
First Published 1999, Delhi Academy of Medical Sciences
© 2021 DAMS Publication

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Contents
Chapter 1 Carbohydrate Chemistry 1-8
Chapter 2 Metabolism of Carbohydrate 9 - 38
Chapter 3 Lipid Chemistry 39 - 46
Chapter 4 Metabolism of Lipid Compounds 47 - 72
Chapter 5 Amino Acid Chemistry 73 - 82
Chapter 6 Amino acid Metabolism 83 - 98
Chapter 7 Protein: Its Various Level of Structure and
Purification 99 - 104
Chapter 8 Enzyme 105 - 114
Chapter 9 Heme Metabolism 115 - 122
Chapter 10 Electron Transport Chain
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Chapter 11 Genetics 131 - 144
Chapter 12 Elementary Genetics 145 - 154
Chapter 13 Genetic Technologies 155 - 164
Chapter 14 Micronutrients (Vitamins and Mineral) 165 - 180
Chapter 15 Nutrition and Energy Metabolism 181 - 190
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1 Carbohydrate Chemistry
CONCEPTS
Â Concept 1.1: Definition & Classification of
carbohydrate
Â Concept 1.2: AfraTafreeh.com
Isomers of carbohydrate
2 | Biochemistry
Concept 1.1: Definition & Classification of Carbohydrate
Learning Objective: At the end of this page learner should be able to
1) Define carbohydrate
2) Classify carbohydrate
Time Needed
1 Reading
st
30 mins
2 Reading
nd
15 mins
Concept Summary:
Carbohydrates are aldehyde/ketone derivatives of polyhydric alcohols. Their general
formula is CnH2nOn. Carbohydrates are broadly classified as simple or complex
carbohydrates.
Simple carbohydrates can further be divided into monosaccharide, disaccharide,

oligosaccharide and polysaccharide.
1. Monosaccharides : Are simple form of carbohydrates which cannot be further
hydrolyzed into simpler carbohydrates. Monosaccharides are represented by
formula CnH2nOn
2. Disaccharides : They yield two molecules of same / different monosaccharide on
hydrolysis. Disaccharides are represented by formula Cn(H2O) n-1.
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3. Oligosaccharides: yield 3-10 molecules of monosaccharide units on hydrolysis.
4. Polysaccharides: yield more than 10 molecules of monosaccharides on
hydrolysis.
Depending on whether polysaccharide has all the monosaccharide similar or different,
they are further divided into homopolysaccharide or heteropolysaccharide.
Homopolysaccharides (Homoglycans): Polymers of same monosaccharides unit.
Further classified into structural and storage homopolysaccharides.
Examples of structural polysaccharides are cellulose, inulin and chitin( exoskeleton
of insects).
Examples of storage polysaccharide as starch and glycogen.
Heteropolysaccharides: Polymers of different monosaccharide units or their
derivatives eg. mucopolysaccharides (glycosaminoglycans), blood group antigen,
Agar, pectin.
High yield points [Direct asked statements]

Simplest carbohydrate Glyceraldehyde
Most abundant carbohydrate D form
Reactive group in fructose Aldehyde
homopolysaccharides Inulin, cellulose, chitin, dextran, starch, glycogen
heteropolysaccharide Mucopolysaccharide, blood group antigen, Agar, Pectin
Carbohydrate Chemistry | 3
Worksheet
• EXTRA POINTS FROM DQB
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4 | Biochemistry
Time to Recall and Analyse
1) Enumerate Homopolysaccharide
2) Enumerate heteropolysaccharide
3) What is chitin?
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Concept 1.2: Isomers of Carbohydrate
1) Define isomers
2) Discuss various types of isomers
Time Needed
1 Reading
st
30 mins
2 Reading
nd
15 mins
Concept Summary:
Compounds which have some structural formula but differ in their physical and
chemical property are known as isomers.
Types of isomers:
A. Optical isomers.
B. Functional isomers.
C. Stereoisomers:
Anomers.
Epimers.
Enantiomers.
Pyranose & furanose ring AfraTafreeh.com
A. Optical isomers: Presence of asymmetric carbon atom confers optical activity on
the compound. When a beam of plane polarized light is passed through a solution
exhibiting optical activity, it will be rotated to right/left (i) if rotated to right, the
compound is called dextrorotatory (d or +sign), when related to left, compound is
called levorotatory (l or-sign).
B. Functional isomer: aldoses and ketoses.
C. Stereoisomers:
a. Anomers: They differ in their spatial orientation of –H and –OH atoms with
regard to first or anomeric carbon atom. Two forms are there – Alpha and Beta
anomers.
Alpha anomers have –OH group below the plane of ring on anomeric carbon
atom.
Beta anomers have –OH group above the plane of ring on anomeric carbon
atom.
b. Epimers: Isomers differing as a result of variation in configuration of the –OH
and –H on carbon atoms 2, 3 and 4 of glucose are known as epimers. e.g.
epimers of glucose are Mannose and galactose formed by epimerization at
carbons 2 and 4, respectively.
c. Enantiomers: D and L forms are enantiomers. Enantiomers are mirror image
of each other. D and L Isomerism: The orientation of H and OH groups around
carbon atom just adjacent to terminal primary alcohol group. (Penultimate
carbon) if – OH group on this carbon atom is towards right, carbohydrate is
called D-isomer, when–OH group is on left, it is a member of L- series.
6 | Biochemistry
D. Pyranose and furanose ring structure – terminology is based upon the fact
that stable ring structure of monosaccharides is similar to ring structure of pyran
or furan.
Racemers: When equal amount of dextrorotatory and levo rotatory isomers are
present the resulting mixture has no optical activity, such a mixture is called
racemic mixture.
Invert sugar: D sucrose is called invert sugar, because it gives L-glucose and
L-fructose by invertase.
Figure Fact:
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Worksheet
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8 | Biochemistry
1) What is the difference in D and L forms
2) What is racemic mixture?
3) What is invert sugar?
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2 Metabolism of Carbohydrate
CONCEPTS
Â Concept 1.1: Glycolysis, PDH complex, TCA cycle
Â Concept 2.2: Gluconeogenesis
HMP Shunt pathway
Â Concept 2.4: Glycogen metabolism and GSD
Â Concept 2.5: Fructose metabolism
Â Concept 2.6: Galactose Metabolism
10 | Biochemistry
Concept 2.1: Glycolysis, PDH complex, TCA cycle
Learning object: At the end of this page learner should be able to
1) Define glycolysis and depict its various steps
2) Differentiate between aerobic and anaerobic glycolysis
3) Describe the rate limiting step of glycolysis and role of insulin
4) Describe fate of pyruvate in mitochondria in aerobic conditions
Time Needed
1 Reading
st
150 mins
2 Reading
nd
100 mins
Concept Summary:
Glycolysis is a cytosolic process which results in ATP production. This process occur
in both aerobic and anaerobic conditions with the formation of pyruvate and lactate
respectively. Total ATP produced in aerobic glycolysis is 9 and in anaerobic is 7 with
net production of 7 and 4 respectively.
Pyruvate produced in aerobic glycolysis is further oxidised in mitosol (mitochondrial
matrix) by PDH complex with gain of 1 NADH(2.5 ATP) into acetyl CoA which is
oxidised in TCA cycle with generation of 10 ATP.
Glucokinase(GK) is type IV isoenzyme of hexokinase(HK) with low affinity and
high Km value for glucose in comparison to hexokinase (180mg/dl vs 0.9 mg/dl
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is the Km value for GK and HK respectively). Rate limiting step of glycolysis is
phosphofructokinase-1(PFK-1) which has Fructose 2,6 bisphosphate as its positive
allosteric modifier. Insulin increases level of Fructose 2,6 bisphosphate and hence
the activity of PFK-1 is enhanced in presence of insulin.

Irreversible steps of glycolysis Glucokinase, Phosphofructokinase, Pyruvate kinase
Steps of substrate level phosphorylation in glycolysis Phosphoglycerate kinase, pyruvate kinase
Total & Net ATP in aerobic glycolysis 9&7
Total & Net ATP in anaerobic glycolysis 4&2
Inhibitors of glycolysis and their target Sodium fluoride: Enolase
Iodoacetate: Glyceraldehyde 3 phosphate dehydrogenase
Arsenate: bypass phosphoglycerate kinase
Three enzymes of PDH complex Pyruvate dehydrogenase
Dihydrolipoyl transacetylase
Dihydrolipoyl dehydrogenase
Five coenzymes needed in TCA cycle Thiamine Pyrophosphate
Lipoic acid
Coenzyme A
FAD
NAD
Enzymes active in dephosphorylated state Glucokinase
Phosphofructokinase
Pyruvate kinase
Pyruvate dehydrogenase
Metabolism of Carbohydrate | 11
Figure fact:
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Steps of glycolysis
12 | Biochemistry
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TCA cycle
Worksheet
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14 | Biochemistry
Label the enzymes and ATP production and utilization at various steps:
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Notes:
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16 | Biochemistry
Concept 2.2: Gluconeogenesis
1) Define gluconeogenesis and Various organs involved in this process
2) Describe various gluconeogenic substrate and steps involved in process of
gluconeogenesis
3) Describe the regulatory enzymes of gluconeogenesis and hormonal action
Time Needed
1 Reading
st
150 mins
2 Reading
nd
100 mins
Concept Summary:
Gluconeogenesis is the process of formation of glucose from non-carbohydrate
substances.
Substrates which are gluconeogenic are mentioned below:
1) Glucogenic amino acid (amino acids that can be converted into glucose),
2) Lactate
3) Pyruvate
4) Propionate
5) Glycerol
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Location: Main organ where gluconeogenesis occurs is the liver and kidney. The
process is partly cytosolic and partly mitochondrial.
Energetics: Synthesis of glucose by the process of gluconeogenesis is energy
consuming process. Conversion of 2 moles of pyruvate into 1 mole of glucose
requires the following:
4 moles of ATP.
2 moles of GTP
2 moles of NADH
This is equivalent of 11 ATP
Steps of formation of glucose from pyruvate are as follows
Key enzymes of gluconeogenesis are
1. Pyruvate carboxylase.
2. Phosphoenol pyrvate carboxykinase.
3. Fructose 1,6 bisphosphatase.
4. Glucose-6-phosphatase.
Hormonal Control on Gluconeogenesis

• Glucocorticids (e.g. cortisol) are the most important stimulating hormones
for gluconeogenesis. These hormones act as inducers of the key hepatic
glucoeogenesis enzymes (i.e. pyruvate carboxylase, phosphoenol pyruvate
carboxykinase, fructose 1,6 bis phosphatase and glucose-6- phosphatase).
• Glucagon and epinephrine also stimulate gluconeogenesis but to a somewhat

lesser extent than cortisol (glucocorticoid).
• Insulin on the other hand is an inhibitor for the key enzymes of gluconeogenesis.
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18 | Biochemistry
Site of gluconeogenesis Partly mitochondrial, partly cytosolic
Energy expenditure for making of one glucose from two pyruvate 11 ATP equivalent
Stages when gluconeogenesis occurs Starvation, diabetes
Most important gluconeogenic amino acid Alanine
Transporter through which glucose is secreted in the blood from GLUT 2
hepatic cell
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Worksheet
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20 | Biochemistry
Label the enzymes at various steps:
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Notes:
Concept 2.3: HMP Shunt Pathway
1) Define HMP shunt pathway and depict its various steps
2) Differentiate between oxidative and nonoxidative phase
3) Describe usage of this pathway
Time Needed
1 Reading
st
150 mins
2 Reading
nd
100 mins
Concept Summary:
HMP shunt pathway is also known as pentose phosphate pathway. It is a multicyclic
process in which 3 molecules of glucose-6-phosphate give rise to 3 molecules of CO2
and three 5 carbon residues, the latter are rearranged to generate 2 molecules of
glucose-6- phosphate and 1 molecule of glycolytic intermediate glyceraldehyde-3-
phosphate.
The main purpose of HMP Shunt pathway is as follow:

1. Generation of NADPH for reductive biosynthesis:
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Following processes are reductive biosynthesis which require NADPH as donor of
reducing equivalent from NADPH.
a) Fatty acid synthesis.
b) Steroid hormone synthesis
c) Cholesterol synthesis
d) Bile acid synthesis
2. Provides a source of Ribose-5- phosphate for nucleic acid biosynthesis.
Location of HMP Shunt pathway:
Erythrocytes, liver, lactating mammary gland, adipose tissue, adrenal cortex.
All the steps of HMP shunt pathway are cytosolic.
Erythrocytes depends on PPP for NADPH which is required to maintain
glutathione in reduced state which is essential to maintain integrity of RBC
membrane
All the Reactions of Pentose phosphate pathway can be divided in 2 Phases
(1) Oxidative / Irreversible phase
(2) Non-oxidative / Reversible phase.
Oxidative phase generates NADPH and nonoxidative phase generates ribose-
5-phosphate.
22 | Biochemistry
Rate limiting step of HMP Shunt pathway G6PD [Glucose 6 phosphate dehydrogenase]
NADPH is produced in Irreversible phase of HMP shunt pathway
Ribose 5 phosphate is produced in Reversible phase of HMP shunt pathway
Intermediates of reversible phase Ribose 5 phosphate
Ribulose 5 phosphate
Xylulose 5 phosphate
Sedoheptulose 7 phosphate
Fructose 6 phosphate
Glyceraldehyde 3 phosphate
Erythrose 4 phosphate
Figure fact:
Steps of oxidative and nonoxidative phases of HMP shunt pathway is depicted
below:
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24 | Biochemistry
Worksheet
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Label the enzymes and ATP production and utilization at various steps:
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Notes:
26 | Biochemistry
Concept 2.4: Glycogen metabolism and GSD
(Glycogen Storage Disorder)
1) Define glycogenesis and depict its various steps
2) Define glycogenolysis and depict its various step
3) Differentiate between glycogenolysis in liver and muscle
4) Describe the rate limiting step of glycogenesis and glycogenolysis
5) Describe various glycogen storage disorder
Time Needed
1 Reading
st
150 mins
2 Reading
nd
100 mins
Concept Summary:
Glycogen is the storage form of carbohydrate which is broken to release glucose
at the time of need. Liver and skeletal muscle are two main organs which store
glycogen. Glycogenesis occurs in well fed state when adequate glucose and insulin
is available. UDP glucose acts as a donor of glucose for this process of glycogenesis.
Rate limiting step is catalyzed by glycogen synthase enzyme which is active in
dephosphorylated form. AfraTafreeh.com
Glycogenolysis occurs in presence of glucagon, epinephrine and norepinephrine.
Rate limiting step of glycogenolysis is glycogen phosphorylase. Glucose 1 phosphate
released by action of glycogen phosphorylase is converted to glucose 6 phosphate
y phosphoglucomutase enzyme. Glucose 6 phosphate either is converted to free
glucose by glucose 6 phosphatase enzyme in liver which enters the blood or this
glucose 6 phosphate is utilized in the muscle for glycolysis and hence energy
production.
Deficiency of various enzyme in glycogen metabolism results in occurrence of various

glycogen storage disorder with manifestation ranging from hypoglycemia to exercise
intolerance. They are summarized in table below:
Glycogenosis Name Cause of disorder Characteristics
Type Ia Von Gierke’s Deficiency of glu-6-phosphatase Hypoglycemia lacticacidemia,

disease ketosis, hyperlipemia.
Type Ib –– Endoplasmic reticlum glucose As type Ia; neutropenia and

6-phosphate transporter recurrent infection.
Type II Pompe’s Deficiency of lysosomal α-1,4 and Fatal, accumulation of glycogen in

disease α-1,6 glucosidase lysosomes, heart failure.
Type IIIa Cori’s disease Absence of debranch Accumulation of characteristic

Type IIIb Cori’s disease Liner along and Muscle branched polysaccharide.
Type IV Andersen’s Absence of branching enzyme Death due to cardiac or liver

disease failure in first year of life.
Type V McArdle’s Absence of muscle phosphorylase Diminished exercise tolerance;

syndrome muscles have abnormally high
glycogen content.
Type VI Her’s disease Deficiency of liver phosphorylase High glycogen content in liver,
tendency towards hypoglycemia.
Type VII Tarui’s disease Deficiency of in msl and RBC As in type V.

phosphofructokinase
Type VIII –– Deficiency of liver Hepatomegaly.

phosphorylasekinase
Type IX –– Deficiency of liver and muscle Hepatomegaly.

phosphorylasekinase
Type X –– cAMP dependent protein kinese A Hepatomegaly.

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Rate limiting step of glycogenesis Glycogen synthase
Rate limiting step of glycogenolysis Glycogen phosphorylase
Active form of glycogen synthase Dephosphorylated form
Active form of glycogen phosphorylase Phosphorylated form
Debranching enzyme will act on Alpha-1,6 linkage to liberate a free glucose residue.
( and not glucose 1-phosphate)
Phosphorylase enzyme specifically acts on the Terminal alpha 1,4 glycosidic bonds of glycogen
molecules resulting in liberation of glucose units as
glucose 1 phosphate
In liver glycogen is 4% and in muscle it is 0.7%, In liver, total stored glycogen is 72 gms, while in
muscle it is 245 gms
28 | Biochemistry
Figure fact:
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Worksheet
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30 | Biochemistry
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Notes:
Concept 2.5: Fructose metabolism
1) Describe fructose metabolism and depict its various steps
2) Discuss various enzyme deficiency associated with fructose metabolism and resulting
consequence
Time Needed
1 Reading
st
150 mins
2nd Reading 100 mins
Concept Summary:
Main organ involved in metabolism of fructose is liver. The process of fructose
metabolism is cytosolic. Fructokinase convert fructose in to Fructose 1 phosphate
and aldolase B acts on fructose 1 phosphate to cleave into DHAP and glyceraldehyde.
Enzyme triokinase convert glyceraldehyde to glyceraldehyde 3 phosphate which
is further metabolized in glycolysis.Deficiency of fructokinase results in benign
fructosuria which is a benign condition and deficiency of enzyme aldolase B
results in hereditary fructose intolerance which is characterized by hepatomegaly,
hypoglycemia, lactic acidosis with accompanying hyperuricemia.
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Type of Aldolase involved in fructose metabolism Aldolase B
Essential fructosuria enzyme deficiency fructokinase
hereditary fructose intolerance enzyme deficiency Aldolase B deficiency
Clinical feature of hereditary fructose intolerance Hepatomegaly
Hypoglycemia
Lactic acidosis
Hyperuricemia
Aversion to sweet food
Figure fact:
32 | Biochemistry
Worksheet
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Draw the main step and enzymes involved in fructose metabolism
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Notes:
34 | Biochemistry
Concept 2.6: Galactose Metabolism
1) Describe galactose metabolism and depict its various steps
2) Discuss various enzyme deficiency associated with galactose metabolism and
resulting consequence
Time Needed
1 Reading
st
150 mins
2nd Reading 100 mins
Concept Summary:
Galactose is metabolized in liver with the help of certain enzyme which convert it finally to
glycolytic intermediate glucose 6 phosphate. The enzymes required are
1) Galactokinase
2) Galactose 1 phosphate uridyl transferase
3) Epimerase
The whole pathway is diagrammatically represented below.

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Deficiency of either of the three enzymes results in Galactosaemia, a condition

which is characterizedd by inability to metabolize dietary galactose.
Galactose-1-phosphate uridyl transferase deficiency leads to classical galactosemia.
This results in increased level of galactose in blood and urine leading to cataract,
mental disturbance, lethargy, vomiting, liver enlargement.
Deficiency of galactokinase results in benign galactosemia which is characterized
by presence of just cataract. No organomegaly and no mental retardation exist in
benign galactosemia.

Galactose is Dietary nonessential
benign galactosemia Deficiency of galactokinase
classical galactosemia Galactose-1-phosphate uridyl transferase deficiency
Cataract seen in Both benign and classical galactosemia
Treatment for galactosemia Lactose and galactose free diet
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36 | Biochemistry
Worksheet
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38 | Biochemistry
Notes:
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3 Lipid Chemistry
CONCEPTS
Â Concept 3.1: Classification of lipid compounds
Description of phospholipid and
glycolipid
40 | Biochemistry
Concept 3.1: Classification of lipid and description of phospholipid
and sphingolipid
a) Define lipids
b) Enumerate the members in each class of lipid classification
c) Describe various phospholipid
d) Describe various sphingolipid and sphingolipidosis
Time Needed
1 Reading
st
30 mins
2 Reading
nd
20 mins
Concept Summary:
The lipids are heterogenous group of compounds related by their physical rather
than by their chemical properties. They have the common property of being:
1) relatively insoluble in water and.
2) soluble in nonpolar solvents such as ether, chloroform and benzene.
They are classified as per modified Bloor classification as follow

1. Simple lipids: Esters of fatty acids with various alcohols. Depending on alcohol
they are of two types:
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a. Fats: Esters of fatty acids with glycerol. A fat in the liquid state is known as oil.
b. Waxes: Esters of fatty acids with higher molecular weight monohydric alcohols
like sphingosine
2. Complex lipids: Esters of fatty acids containing “groups” in addition to an alcohol
and fatty acids.
a. Phospholipids: Lipids containing fatty acid, alcohol and a phosphoric acid
residue. Eg. glycerophospholipids and sphingophospholipids.
b. Sphingolipids: Lipids containing a fatty acid, sphingosine, and carbohydrates.
Can be divided further in to sphingophospholipid and sphingoglycolipid.
c. Other Complex Lipids: for eg. sulfolipids, aminolipids and lipoproteins.
3. Precursor and derived lipids: These include fatty acids, glycerol, steroids, alcohol
etc.
Phospholipid:
Phospholipids are major constituents of plasma membrane.
Classification of Phospholipid
1. Phosphatidycholine (lecithin).
2. Phosphatidylethanolamine (a cephalin).
3. Phosphatidylserine.
4. Phosphatidylinositol.
5. Cardiolipin (major lipids of mitochondrial membrane).
Sphingolipids
Sphingolipids are found in central nervous system and specially in white
matter.
a. Sphingomyelin: Sphingomyelin on hydrolysis yields a fatty acid, phosphoric
acid, choline and a complex amino alcohol, sphingosine.
The combination of sphingosine plus fatty acids is known as ceramide.
b. Glycosphingolipids: Sphingolipids that contain carbohydrates moieties.
Cerebrosides are ceramide monohexosides (e.g. galactocerebroside and
glucocerebroside).
Sulfatides are cerebrosides that contain sulphated sugars.
β-sulfogalactocerebroside.
Globosides are ceramide oligosaccharides that contain two or more sugar
molecules, most often galactose, glucose or N-acetylgalactosamine, attached
to ceramide.
Gangliosides are glycosphingolipids that contain one or more neuraminic acid
residues, usually N-acetyl derivative, which is sallic acids.
Sphingolipidoses:
Sphingolipidoses are inherited genetic disorder referred to as lipid storage diseases,
in which there is deficiency of an enzyme involved in in the normal catabolism,
particular of sphingolipids.
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Disease Enzyme Deficiency Lipid Accumulating1
Fucosidosis α-Fucosidase Cer–Glc–Gal–GalNAc–Gal–Fuc
H-Isoantigen
Generalized gangliosidosis GM1-β-galactosidase Cer–Gio–Gal (NeuAc)–GalNAo–Gal
GM1 Ganglioside
Tay-Sachs disease Hexosaminidase A Cer–Glc–Gai(NeuAc)–GalNAc
GM2 Ganghoside
Tay-Sachs variant or Sandhoff's Hexosaminidase A and B Cer–Gio–Gal–Gal–GalNAc
disease Globoside plus GM2 ganglioside
Fabry's disease α-Galactosidase Cer–Gic–Gal–Gal
Globotriaosylceramide
Caramide lactoside lipidosis Ceramide lactosidase Cer–Glo–Gal
(β-galactosidase) Ceramide lactoside
Metachromatic * Arylsuifataso A Cer–Gal–OSO3
leukodystrophy 3-Sulfogalactosylceramide
Krabbe's disease β-Galactosidase Cer–Gal
Galactosylceramide
Gaucher's disease β-Glucosidase Cer–Glc
Glucosylceramide
Niemann-Pick disease Spingomyelinase Cer–P–choline
Sphingomyelin
Farber's disease Ceramidase Acy–Sphingosine
Ceramide
42 | Biochemistry
Figure facts
Phosphatidylcholine Phosphatidic acid
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Phosphatidylethanolamine
Phosphatidylinositol
Lysophosphatidylcholine (Lysolecithin)
Plasmalogen (Phosphatidylethanolamine)
A sphingomyelin
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Phosphatidycholine is also called as Lecithin
Most abundant phospholipid is Lecithin
Phosphatidylethanolamine is also called as Cephalin
Phospholipid as precursor of second messenger Phosphatidylinositol
Ceramide is combination of sphingosine plus fatty acids is known
as ceramide
Cerebroside are Ceramide monohexosides
Gangliosides are glycosphingolipids that contain one or more
neuraminic acid residues, usually N-acetyl
derivative, which is sallic acids
44 | Biochemistry
Worksheet
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Identify the base in this phospholipid
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46 | Biochemistry
Notes:
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4 Metabolism of
Lipid Compounds
CONCEPTS
Â Concept 4.1: F
atty acid and its metabolism
(Synthesis and oxidation)
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Â Concept 4.2: V
arious lipoproteins and their
metabolism
Â Concept 4.3: C
holesterol metabolism and Bile
acid
48 | Biochemistry
Concept 4.1: Fatty acid Synthesis
a) Describe the steps of fatty acid synthesis
b) Describe fatty acid chain elongation and desaturation
Time Needed
1 Reading
st
120 mins
2 Reading
nd
80 mins
Concept Summary:
Fatty Acid Synthesis:
De Novo Synthesis:
This system is present in the soluble (cytosol) fraction of cells in many tissues e.g.
liver, kidney, brain, lung, mammary gland and adipose tissue.
• Its cofactor requirements include NADPH, ATP, Mn2+, and HCO3–.
• Acetyl CoA is the starting substrate.
How acetyl CoA synthesizes fatty acid:

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Fatty acid synthesis depends on a cytoplasmic supply of acetyl CoA.
• Glucose is catabolized to acetyl CoA which combines with oxaloacetate to form
citrate.
• Amino acids are also degraded to compounds that can enter the citric acid cycle
to form citrate.
• Citrate is then transported across the mitochondrial membrane to the cytoplasm,
where it is cleaved by citrate lyase enzyme to form oxaloacetate and acetyl CoA.
Acetyl CoA Carboxylase:
It is the rate limiting enzyme for synthesis of fatty acid.
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Subsequently fatty acid synthase complex is required, which is multienzyme complex.

Fatty acid synthase complex: It is cytosolic complex which is a dimer 2 units of polypeptide
one arranged in head to tail lantiguration.
50 | Biochemistry
Sourse of NADPH for fatty acid:
1. HMP shunt. 2. Malic enzyme. 3. Isocitrate dehydrogenase.
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Fatty acid chain elongation and denaturation:

Microsomal elongase system:
1. Present in ER.
2. Uses malonyl CoA.
3. Adds 2 carbon units in each cycle.
4. Requires NADPH.
5. Elongation occurs at COOH terminal.
Microsomal desaturase system:
1. Mammals have only Δ9, Δ6, Δ5, Δ4 desaturase.
2. ω 6 and ω 3 family are dietary essential.

Mammals can synthesise only even chain fatty acid cofactor requirements for fatty acid synthesis include
NADPH, ATP, Mn2+, and HCO3–.
Acetyl CoA is the starting substrate for synthesis of fatty acid
Acetyl co A carboxylase is the rate limiting enzyme for synthesis of fatty acid.
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Fatty acid synthase complex: It is cytosolic complex which is a dimer 2 units of polypeptide one arranged
in head to tail configuartion
52 | Biochemistry
Worksheet
 AfraTafreeh.com

Draw the diagram denoting the rate limiting enzyme of fatty acid synthesis
Notes:
AfraTafreeh.com
54 | Biochemistry
Concept 4.2: Fatty Acid Oxidation and Ketone Body Metabolism
1) Describe various types of fatty acid oxidation
2) Discuss ketone body synthesis
3) Discuss ketone body utilization
Time Needed
1 Reading
st
120 mins
2 Reading
nd
80 mins
Concept Summary:
Fatty Acid Oxidation:
Oxidation of fatty acids generates the high- energy compounds reduced NAD (NADH)
and reduced flavin adenine dinucleotide (FADH2) and yields acetyl CoA.
In beta-oxidation two carbons are cleaved at a time from acyl-CoA molecules,
starting at the carboxyl end.
Role of Carnitine:
Synthesized from lysine + Methionine. Long chain fatty acyl Co A cannot freely
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diffuse across the inner mitochondrial membrane. Carnitine in the inner mitochondrial
membrane mediates transfer of fatty acyl groups from the cytosol to the mitochondrial
matrix where they are oxidized.
Once acyl CoA enters the mitochondrial matrix, it undergoes â oxidation and it is a
cyclical process involving steps depicted in the figure.
As can be seen in above figure, each cycle generates 1 FADH2 and 1 NADH. Completion
of each cycle results in removal of 2 carbon moiety acetyl CoA at a time. This acetyl
CoA enters TCA cycle where it generates 10 ATP.
Stoichiometry of Beta Oxidation:

Net ATP produced from 1 molecule of palmitic acid will be 108-2=106.
7 NADH 7 × 2.5 = 17.5 ATP
7 FADH2 7 × 1.5 = 10.5 ATP
8 Acetyl CoA 8 × 10 = 80 ATP
Total produced 108 ATP
Oxidation of Fatty acids that have an odd number of carbon atoms:
1. They undergo â-oxidation as acyl CoA derivatives until a three-carbon fragment,
propionyl CoA, is formed.
2. Propionyl CoA is carboxylated to methylmalonyl CoA by biotin-dependent propionyl
CoA carboxylase.
3. Methylmalonyl CoA is converted to succinyl CoA by methylmalonyl CoA mutase.

4. Succinyl CoA can be metabolized via the citric acid cycle, of which it is an
intermediate.
á- and ù oxidation of fatty acids.
á-oxidation:
Removal of one carbon at a time from the carboxyl end of the molecule.
Has been detected in brain tissue.
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56 | Biochemistry
Alpha oxidation, is normally a minor pathway and is brought about by hydroxylase

enzymes involving cytochrome P450 in the endoplasmic reticulum. It produces
dicarboxylic acid as –CH3 group is converted to –COOH group.
Ketone body and their metabolism

These are small, water soluble, energy yielding molecules, produced mainly by liver
along with a small amount produced in the kidneys.
These are:
1. Acetoacetate.
2. â - hydroxy butyrate.
3. Acetone.
Ketone bodies are the preferred energy substrates for the heart, skeletal muscle and
kidney during fasting state.
Some salient features of ketone bodies:
1. â- hydroxy butyrate is the most abundant ketone body under normal physiological
state.
2. â- hydroxy butyrate is the only ketone body having no keto group.
3. Acetone is a ketone body, which gets volatilized along with exhaled air and thus,
can’t be used as a fuel for tissues.
4. â- hydroxy butyrate yields more energy then acetoacetete.
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5. Liver synthesizes ketone bodies but is unable to utilize these, as it lacks the
enzyme thiophorase.
6. Nervous tissue, which obtains almost all of its energy from glucose normally, is
unable to take up and use fatty acids bound to albumin for energy purpose, as
fatty acids can’t cross the blood brain barrier. However, it can use ketone bodies
when glucose supplies are insufficient
Ketone body synthesis:

Following are the steps of ketone body synthesis
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Ketone Body Utilization:

Ketone bodies are mainly utilized in following organs:
• Muscle
• Heart.
• Brain.
58 | Biochemistry
For utilization ketone body is converted to acetoacetyl CoA , for which succinyl Co A is
the donor of Co A moiety in presence of enzyme thiophorase.
Liver does not utilize ketone body due to absence of the enzyme thiophorase.
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Beta oxidation occurs in Mitochondrial matrix
Carnitine shuttle uses 2 ATP
Ketogenesis is Mitochondrial matrix of liver
Ketolysis is Mitochondrial matrix of extrahepatic cells
Palmitic acid complete beta oxidation 108 ATP TOTAL
Palmitic acid complete beta oxidation net 106 ATP NET
Worksheet
 AfraTafreeh.com

60 | Biochemistry
Calculate the total and net ATP produced after complete beta oxidation of
palmitic acid
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Notes:
Concept 4.3: Various lipoproteins and their Metabolism
Learning objective: At the end of this page learner should be able to
1) Describe various lipoprotein and their metabolism
2) Discuss various hyper lipoproteinemias
Time Needed
1 Reading
st
120 mins
2 Reading
nd
80 mins
Concept Summary:
A typical lipoprotein consists of a lipid core of mainly nonpolar triacylglycerol
and cholesterol ester surrounded by a surface layer of more polar phospholipid,
cholesterol and the protein fraction known as apolipoprotein or apoprotein.
Major classes of lipoprotein are:
1. Chylomicrons which transport dietary (exogenous) triglycerides of intestine to
blood.
2. Very low density lipoproteins (VLDL or pre-â-lipoproteins), transporting
endogenous triacylglycerol from liver to blood. It also contains lesser amount of
cholesterol.
3. Low density lipoproteins (LDL or â-lipoproteins) with represents the final stage
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in catabolism of VLDL and transport plasma cholesterol from liver to tissues.
4. High density lipoproteins (HDL or á-lipoproteins) which transport plasma
cholesterol from tissue to liver. HDL particles are also involved in VLDL and
chylomicrons metabolism.
Triacylglycerol is the predominant lipid in chylomicrons and VLDL.
Whereas cholesterol and phospholipids are predominant lipids in LDL and HDL
respectively.
62 | Biochemistry
Chylomicron Metabolism:
Following diagram explain the steps of chylomicron metabolism:
Metabolic fate of chylomicrons. (A, apolipoprotein A; B-48, apolipoprotein B-48; apolipoprotein C; E,

apolipoprotein E; HDL, high-densuty lipoprotein: TG, triacylgycerol: C, cholesterol and cholesteryl ester;
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P, phospholipid; HL hepatic lipase.) Only the predominant lipids are shown
VLDL Metabolism:
Following diagram explain the steps of VLDL metabolism:
Metabolic fate of VLDL (A, apolipoprotein A; B-48, apolipoprotein B-48; apolipoprotein C; E, apolipoprotein
E; HDL, high-densuty lipoprotein: TG, triacylgycerol: C, cholesterol and cholesteryl ester; P, phospholipid; HL
hepatic lipase.)Only the predominant lipids are shown
Fate of LDL Cholesterol at the Tissue Level:
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Metabolic fate of LDL
Reverse Cholesterol Transport and HDL Cycle:

64 | Biochemistry
Hyperlipoproteinemias:
Various hyperlipoproteinemias are described in table below
Type Generic Classification Increased Apoprotein Increased Lipid
I Lipoprotein lipase deficiency Chylomicrons Triacylgycerols
IIa Hypercholesterolemia LDL Cholesterol

(LDL receptor deficiency)
IIb Combined hyperlipidemia LDL, VLDL Triacylglycerols,

Cholesterol
III Dysbetalipoproteinemia IDL and CM Remnant Triacylglycerols,

(Remnant Removal disease) Cholesterol
IV Hypertriglyceridemia VLDL Triacylglycerols
V Mixed hyperlipidemia VLDL, chylomicrons Triacylglycerols

Largest lipoprotein Chylomicron
Lipoprotein with least density Chylomicron
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Lipoprotein with least mobility on electrophoresis
Chylomicron
Lipoprotein with maximum lipid content Chylomicron
LDL receptor mutation results in Type II hyperlipoproteinemia
Worksheet
 AfraTafreeh.com

66 | Biochemistry
Mention all dyslipoproteinemias.
Notes:
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Concept 4.4: Cholesterol metabolism and Bile acid
1) Describe Metabolism of Cholesterol
2) Describe Bile Acid Synthesis
Time Needed
1 Reading
st
120 mins
2 Reading
nd
80 mins
Concept Summary:
Cholesterol Metabolism:
Cholesterol is the precursor of the steroid hormone, vitamin D and bile salts.
A little more than half of body cholesterol arises by synthesis and remainder is
provided by average diet.
The liver is the major site of cholesterol biosynthesis. The microsomal (endoplasmic
reticulum) and cytosol fraction of cell is responsible for cholesterol synthesis.
Cholesterol Biosynthesis:
1. 3-Hydroxy-3-methylglutaryl CoA (HMG CoA) is formed in the cytosol from acetyl
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CoA in two steps by thiolase and HMG CoA synthase.
2. HMG CoA to converted to mevalonate by HMG CoA reductase, an NADPH-
dependent enzyme. This is the key regulatory site of cholesterol biosynthesis.
This is regarded as the rate-limiting step in cholesterol biosynthesis.
The feeding of cholesterol reduces the hepatic biosynthesis of cholesterol by
reducing the activity of HMG CoA reductase.
HMG CoA is also reduced by fasting which limits the availability of acetyl CoA
and NADPH.
68 | Biochemistry
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Hormonal Effects on Cholesterol Biosynthesis:

• Insulin: Stimulates HMG CoA reductase activity.
• Glucagon: Antagonizes the effect of insulin.
• Thyroid hormone stimulates HMG CoA reductase activity.
Drugs:
1. Statin group of drugs blocks endogenous cholesterol synthesis by inhibiting HMG CoA
reductase activity.
2. Mevalonate is activated with high- energy phosphate bonds and then decarboxylated
to form five carbon isoprenoid isomers.
3. Condensation of isoprenoid units forms squalene then lanosterol, zymosterol,
desmosterol and finally cholesterol.
Bile Acids:
Primary bile acids: Cholic and chenodeoxycholic acids are formed in the liver from
cholesterol.
Secondary bile acids: Deoxycholic acid and lithocholic acid are formed from
primary bile acids in the intestine through the action of intestinal bacterial enzymes.
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1. 7〈-hydroxylation of cholesterol is the first committed step in the biosynthesis

of bile acids and this is also a rate limiting step. 7〈-hydroxylase a microsomal
enzymes requires oxygen NADPH and cytochrome P450 and Vitamin C deficiency
interferes with bile acids formation at the 7〈-hydroxylation steps.
2. Primary bile acids enter the bile as glycine or taurine conjugates. Since bile
contains significant quantities of sodium and potassium and the pH is alkaline, so
bile acid and their conjugates are in salt from—hence the term “bile salts”.
3. A portion of the primary bile acids in the intestine is subjected to further changes
by the activity of the intestinal bacteria. These include deconjugation and
7〈-dehydroxylation, which produce the secondary bile acids, deoxycholic acid and
lithocholic acid.
70 | Biochemistry
The primary and secondary bile acids are absorbed almost exclusively in the ileum,
returning to liver by way of portal circulation. Which is 98-99% of bile acids secreted
into the intestine. This is known as enterohepatic circulation

Cholesterol is synthesized from Acetyl CoA
Rate limiting step of cholesterol synthesis is HMG CoA reductase
Statin inhibits HMG CoA reductase
Rate limiting step of bile acid synthesis is 7 alpha hydroxylase
Primary bile acid is Cholic acid, chenodeoxycholic acid
Secondary bile acid is Deoxycholic acid and lithocholic acid
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Worksheet
 AfraTafreeh.com

72 | Biochemistry
1) What is the role of insulin on rate limiting step of cholesterol synthesis?
2) What are the biochemical changes occur in primary bile acid to convert it to secondary
bile acid?
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Notes:
5 Amino Acid Chemistry
CONCEPTS
Â Concept 5.1: Classification of amino acids
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Â Concept 5.2: Amino acid degradation and urea
cycle
74 | Biochemistry
Concept 5.1: Classification of amino acids
1) Define amino acid structure
2) Classify amino acids based on its different criteria
Time Needed
1 Reading
st
00 mins
2 Reading
nd
00 mins
CONCEPT SUMMARY:
Common amino acids have a general structure. They contain a central alpha (〈)
carbon atom to which a carboxylic group, an amino group, a hydrogen atom and in
addition a side chain R are attached
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Amino acids are classified based on their structure, polarity, nutritional requirement,
and metabolic fate.
Classification of amino acids based on structure

A. Aliphatic Amino Acid:
1. Monoamino monocarboxylic acid: Simple: glycine, alanine.
Branched: valine, leucine, isoleucine.
Hydroxyl: serine, threonine.
Sulphur containing: cysteine, methionine.
Amide group containing: glutamine, asparagine.
2. Monoamino dicarboxylic acid: aspartic acid, glutamic acid.
3. Dibasic monocarboxylic acid: arginine, lysine.
B. Aromatic amino acid: phenylalanine, tyrosine.
C. Heterocyclic amino acid: histidine, tryptophan.
D Imino amino acid: proline.
E Derived amino acid: hydroxyproline, hydroxylysine, ornithine, citrulline.
Amino Acid Chemistry | 75
Classification of amino acid based on polarity of side chain

• Nonpolar: . Phenylalanine, proline, tyrosine, tryptophan ,methionine, isoleucine,
leucine, alanine, Valine.
• Polar.
• Uncharged: Glycine, serine, cysteine, threonine
• Acidic: Aspartic acid, glutamic acid.
• Basic: Arginine, Lysine, Histidine.
Classification of amino acid based on metabolic fate

• Ketogenic: leucine.
• Glucogenic and ketogenic: PITT-L (phenylalanine, isoleucine, tyrosine,
tryptophan, lysine).
• Purely glucogenic: rest 14 amino acids are purely glucogenic.
Classification of amino acid based on dietary need

Essential: Arginine, histidine, isoleucine, leucine, threonine, lysine, methionine,
Phenylalanine, tryptophan, valine.
Remaining ten amino acids are nonessential
Characteristic of peptide bond

• Partially double bond AfraTafreeh.com
• Trans
• Rigid
• Uncharged but polar

Imino acid is Proline
Acidic amino acid Glutamic acid
Aspartic acid
Most basic amino acid Arginine
Least basic amino acid histidine
Tryptophan is Dietary essential
Tyrosine is Dietary nonessential
76 | Biochemistry
Worksheet
 AfraTafreeh.com

Notes: AfraTafreeh.com
78 | Biochemistry
Concept 5.2: Amino acid degradation and urea cycle
1) Describe transamination and oxidative deamination
2) Describe various steps involved in urea cycle
3) Describe various urea cycle disorder
Time Needed
1 Reading
st
00 mins
2 Reading
nd
00 mins
Concept Summary:
Transamination and oxidative deamination
Transamination involves the transfer of an amino group from an amino acid to an
〈-keto acid to form a new amino acid and new 〈-keto acid. This process involves the
interconversion of a pair of amino acids and a pair of keto acids, catalyzed by a
group of enzymes called aminotransferases.
Amino acids undergo transamination to concentrate nitrogen in glutamate, which is
the only amino acid that undergoes oxidative deamination to a significant extent to
liberate free NH3 for urea synthesis.
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Oxidative deamination is catalysed by glutamate dehydrogenase enzyme found in
mitochondrial matrix
Urea synthesis
Key points
• Urea is synthesized in the liver
• It has two amino groups, one derived from ammonia and the other from aspartate.
Carbon atom is supplied by CO2 (as HCO3–).
• Urea is the major end product of protein catabolism in humans.
• The first two enzymes of urea cycle are present in mitochondria while the rest are
localized in cytosol.
• Synthesis of 1 mole of urea require four moles of ATP
• N-acetyl glutamate functions solely as enzyme activator for carbamoyl phosphate
synthetase.
Urea cycle disorder

1. Type I hyperammonemia is due to a defect in carbamoyl phosphate synthetase.
2. Type II hyperammonemia is due to a defect in ornithine transcarbamoylase.
3. Citrullinemia is due to a defect in arginosuccinate synthetase.
4. Arginosuccinic aciduria is due to a defect in arginosuccinate lyase.
5. Hyperargininemia is due to a defect in arginase.
Figure facts
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Synthesis of one molecule of urea require 3 ATP and 4 high energy phosphate
Glutamate dehydrogenase is found in Mitochondrial matrix of liver
Rate limiting enzyme for urea synthesis is carbamoyl phosphate synthetase I
N-acetyl glutamate is Positive allosteric regulator of CPS I
Urea synthesis is Partly mitochondrial and partly cytosolic process

80 | Biochemistry
Worksheet
 AfraTafreeh.com

Mention the various enzymes needed for urea synthesis and disorder related
with deficiency of these enzymes.
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82 | Biochemistry
Notes:
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6 Amino acid Metabolism
CONCEPTS
Â Concept 6.1: Amino acid degradation and urea
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cycle
Â Concept 6.2: Simple and branched chain amino
acid metabolism
Â Concept 6.3: Aromatic amino acid metabolism
Â Concept 6.4: Sulphur containing amino acid
84 | Biochemistry
Concept 6.1: Simple and branched chain amino acid metabolism
a) Describe important points related to glycine
b) Describe important points related to branched chain amino acid
Time Needed
1 Reading
st
00 mins
2 Reading
nd
00 mins
Concept Summary:
GLYCINE
• Smallest amino acid.
• Nonessential.
• Optically inactive.
• Polar.
• Glycogenic.
Important compound where glycine is required

1. AfraTafreeh.com
Purines [C4, C5 and N7 atoms].
2. Glutathione.
3. Heme.
4. Creatine (Glycine + Arginine + Methionine) (GAM).
5. Conjugation with bile acid.
6. Glycine + benzoic acid produce hippuric acid.
BRANCHED CHAIN AMINO ACID

Branched chain amino acid
• Valine – Essential, Glycogenic.
• Leucine – Essential, Ketogenic.
• Isoleucine – Glycogenic + Ketogenic, Essential.
The first three metabolic reactions are common to these three amino acids:
• Transamination.
• Oxidative decarboxylation.
• Dehydrogenation.
Amino Acid Metabolism | 85
Diseases associated with deranged metabolism of branched

chain amino acid
A. Maple Syrup Urine Disease: ( MSUD): This disease is characterized by burnt
sugar odor of urine and is due to defect in the enzyme α-keto acid dehydrogenase.
B. Isovaleric acidemia: Defect is in isovaleryl CoA dehydrogenase. Hence only
leucine catabolism is affected. In urine, isovaleryl CoA is excreted and urine gives
cheesy odor.

Glycine donates atom in purine ring C4, C5 and N7 atoms
Creatine synthesis require Glycine + Arginine + Methionine
Hippuric acid is made up of Glycine + benzoic acid
Maple Syrup Urine Disease: (MSUD) enzyme deficiency α-keto acid dehydrogenase
Maple Syrup Urine Disease: ( MSUD) : urine odor Burnt sugar odor
Isovaleric acidemia: enzyme deficiency Isovaleryl CoA dehydrogenase
Isovaleric acidemia: urine odor Cheesy odor
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86 | Biochemistry
Worksheet
 AfraTafreeh.com

88 | Biochemistry
Concept 6.2: Aromatic Amino Acid Metabolism
Time Needed
1st Reading 00 mins
2 Reading
nd
00 mins
Concept Summary:
Phenylalanine, tyrosine and tryptophan are aromatic amino acid. Phenylalanine is converted
to tyrosine by hydroxylation reaction where tetrahydrobiopterin is the donor of reducing
equivalent.
• Screening test for phenylketonuria: FeCl3 test.
• Confirmatory test for phenylketonuria: Guthrie test.
Tyrosine is responsible for the synthesis of a variety of important products:

• Melanin.
• Catecholamine.
• Thyroid hormone.
• Tyramine. AfraTafreeh.com
Figure facts
Hormone
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As phenylalanine is converted to tyrosine, a single pathway is responsible for the

degradation of both these amino acids, which occurs mostly in liver
90 | Biochemistry
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Tryptophan metabolism
Key points regarding tryptophan
• Essential.
• Both glucogenic and ketogenic.
• Contains an indole ring.
• Metabolism of tryptophan is divided into:
a. Kynurenine pathway.
b. Serotonin pathway.
1. K
ynureninase is a Vit B6 (PLP) dependent enzyme. Deficiency of VitB6 results
in partial failure to catabolize kynurenine, which then forms Xantheurenic
acid.
2. H
artrup’s disease: It is due to an impairment in the absorption and/or
transport of tryptophan and other neutral amino acids from intestine and
renal tubules.
Low levels of these amino acids in plasma and elevated urinary excretion

Classical phenylketonuria Enzyme deficiency is phenylalanine hydroxylase
Tyrosinemia type II Enzyme deficiency is tyrosine transaminase
Tyrosinemia type I Enzyme deficiency is hydrolase
alkaptonuria Enzyme deficiency is homogentisic acid oxidase
albinism Enzyme deficiency is tyrosinase
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Rate limiting step for catecholamine synthesis
Tyrosine hydroxylase
Test to detect indole in urine Obermeyer test

92 | Biochemistry
Worksheet
 AfraTafreeh.com

Enumerate the diseases involved in tyrosine metabolic defect:
94 | Biochemistry
Concept 6.3: Sulphur containing amino acid
Describe the key points of sulphur containing amino acids
Time Needed
1 Reading
st
00 mins
2nd Reading 00 mins
Concept Summary:
Sulphur containing amino acid are
a) Methionine
b) Cysteine
Key points regarding methionine

• Essential amino acid.
• Serves as a precursor for the synthesis of cysteine and cystine.
• It is also required for the initiation of protein biosynthesis. It is the initiator codon
for eukaryotic translation.
Sulphur in methionine is present in thioether linkage (C-S-C), which is very stable
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Specialized product of methionine
1) SAM (S-Adenosyl Methionine).
2) Creatine.
SAM is highly reactive and transfers the methyl group to an acceptor
Metabolic defect in Sulphur containing amino acid metabolism
1. Homocystinurias:
Heritable defects of methionine metabolism.
Up to 300 mg of homocystine, together with SAM (sometimes), is excreted
daily in the urine.
Plasma methionine levels are also elevated.
2. Cystinuria (Cystine – lysinuria):
Inherited metabolic disease, characterized by urinary excretion of cystine up
to 30 times normal.
Excretion of lysine, arginine and ornithine also rises, suggesting a defect in the
renal reabsorption mechanisms for these four amino acids.
Since cystine is relatively insoluble, cystine calculi form in the renal tubules of
these patients.
3. Cystinosis – (cystine storage disease):
Rare lysosomal disorder characterized by defective carrier mediated transport
of cystine.
Cystine crystals are deposited in tissues and organs, particularly the RE system.
Usually accompanied by generalized amino aciduria.
Specialized product of methionine SAM (S-Adenosyl Methionine).
Creatine
Methionine is Essential amino acid
Cysteine is Nonessential amino acid
Typical HCU is due to Deficiency of Cystathione beta synthase
Cystathione beta synthase Require vitamin B6
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96 | Biochemistry
Worksheet
 AfraTafreeh.com

AfraTafreeh.com
98 | Biochemistry
Notes:
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7 Protein: Its Various Level of
Structure and Purification
CONCEPTS
Â Concept 7.1: Protein: Its Various Level of
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Structure and Purification
100 | Biochemistry
Concept 7.1: Protein: Its Various Level of Structure and Purification
a) Describe various level of protein structure
b) Mention key points of alpha helix and beta pleated sheet
c) Enumerate various bonds existing in various level of protein structure
Time Needed
1st Reading 20 mins
2 Reading
nd
10 mins
Concept Summary:
Definitions
Primary structure of protein
It denotes number and sequence of amino acid in a polypeptide chain
Secondary structure of protein
It denotes configurational relationship between the amino acid which are 3 to 4
residue apart in the linear chain.
Alpha Helix: AfraTafreeh.com
• Most common and stable form
• Spiral structure
• 3.6 residue of amino acid/turn
• Right handed helix
• Proline never found in the alpha helix
• Glycine is also rare
Beta pleated sheet
• Extended polypeptide
• May be parallel or anti parallel
• Hydrogen bonds are “interchain” and perpendicular
• Glycine is the main amino acid and proline is also common.
• It is a structural motif of the silk fibroin, flavodoxin, carbonic anhydratase
Tertiary structure of protein
• It is a three dimensional structure of the whole protein
Quaternary structure of the protein
• Subunit interaction between different monomers is known as quaternary structure.
This level of structure exists only in those proteins who has more than one subunit
in them.
Protein: Its Various Level of Structure and Purification | 101
Figure facts
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Bond of primary structure Peptide bond
Bond of secondary structure Hydrogen bond
Bond of tertiary structure Hydrogen bond
Hydrophobic bond
Electrostatic (ionic) bond/Salt bridges
Van-der walls forces
Covalent (disulfide)cross link
Bond of quaternary structure Hydrogen bond
Hydrophobic bond
Electrostatic(ionic)bond/Salt bridges
Van-der walls forces
102 | Biochemistry
Worksheet
 AfraTafreeh.com

Protein: Its Various Level of Structure and Purification | 103
Mention the bonds present in tertiary structure of the protein
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104 | Biochemistry
Notes:
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8 Enzyme
CONCEPTS
Â Concept 8.1: ENZYME CLASSIFICATION and
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Mechanism of Action
Â Concept 8.2: Enzyme Inhibitors
106 | Biochemistry
Concept 8.1 : ENZYME CLASSIFICATION and Mechanism of Action
Time Needed
1st Reading 25 mins
2 Reading
nd
10 mins
Concept Summary:
Enzymes are divided into six major classes with several subclasses:
a. Oxidoreductases are involved in oxidation and reduction.
b. Transferases transfer functional groups (e.g., amino or phosphate groups).
c. Hydrolases transfer water; that is, they catalyze the hydrolysis of a substrate.
d. Lyases add (or remove) the elements of water, ammonia or carbon dioxide (CO2)
to (or from) double bonds.
e. Isomerases catalyze rearrangements of atoms within a molecule.
f. Ligases join two molecules
Enzymes increase the rate of reaction by decreasing the energy of activation.
Energy of activation is required to sufficiently energize a substrate molecule to
reach a transition state
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The Michaelis constant is characteristic of an enzyme and a particular substrate and
reflects the affinity of the enzyme for that substrate. Km is numerically equal to the
substrate concentration at which the reaction velocity is equal to ½ Vmax. Km does not
vary with the concentration of enzyme.
Michaelis-Menten Equation. The Michaelis-Menten equation describes how
reaction velocity varies with substrate.
where v0= Initial reaction velocity Vmax = Maximal velocity

Km = Michaelis constant = (k1 + k2)/k1 [S] = Substrate concentration
Lineweaver-Burke plot. When the reaction velocity, v0, is plotted against the
substrate concentration, [S], it is not always possible to determine when Vmax has
been achieved, because of the gradual upward slope of the hyperbolic curve at
high substrate concentration. However, if 1/v0 is plotted vs. 1/[S], a straight line
is obtained. This plot is called the Lineweaver-Burke plot (also called a double-
reciprocal plot) and can be used to calculate km and Vmax as well as well as to
determine
1. The intercept on the x axis is equal to –1/Km.
2. The intercept on the y axis is equal to 1/ Vmax.
Enzyme | 107
Figure facts : diagram to show lineweaver burke plot
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Oxidoreductase belong to Class I enzyme
Kinase belong to Class II
aldolase Class IV
Double reciprocal plot Lineweaver Burk plot
Isoenzymes
ISOZYMES are different molecular forms of enzymes that may be isolated from the
same or different tissue. Isozymes are physically distinct and separable forms of a
given enzyme.
Clinical use. Analysis of the distribution of isozymes of particular enzymes is sometimes
a useful tool in clinical diagnosis.
Coenzyme is a specific, heat stable, low molecular weight organic molecule which is
required in a chemical reaction. Prosthetic group denotes covalently bound coenzyme.
108 | Biochemistry
Worksheet
 AfraTafreeh.com

Enzyme | 109
Calculate the value of Km in this plot
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Notes:
110 | Biochemistry
Concept 8.2: Enzyme Inhibitors
a) Define competitive and noncompetitive inhibitors
b) Mention the effect of competitive and noncompetitive inhibitors on Vmax and Km
Time Needed
1 Reading
st
30 mins
2 Reading
nd
15 mins
Concept Summary:
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 that side
1. Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [S].
At a sufficiently high substrate concentration, the reaction velocity reaches the
Vmax the absence of inhibitor.
2. Effect on Km: A competitive inhibitor increases the apparent Km for a given
substrate. This means that in the presence of a competitive inhibitor more
substrate is needed to achieve ½ Vmax.
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3. Effect on Lineweaver-Burke plot: Competitive inhibition shows a
characteristic Lineweaver-Burke plot in which the plots of the inhibited and
uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is unchanged). The
inhibited and uninhibited reactions show different X-axis intercepts, indicating
that the apparent Km is increased in the presence of the competitive inhibitor.
Malonate as an example of a competitive inhibitor: Succinate dehydrogenase
catalyzes the oxidation of succinate to fumarate. Malonate is structurally similar to
the substrate and competes for binding at the active site of the enzyme.
Enzyme | 111
A. Noncompetitive inhibition. This type of inhibition is recognized by its

characteristic effect on Vmax and occurs when the inhibitor and substrate bind at
different sites on the enzyme. The noncompetitive inhibitor can bind either free
enzyme or the ES complex, thereby preventing the reaction from occurring.
1. Effect on Vmax: Noncompetitive inhibition cannot be overcome by increasing
the concentration of substrate. Thus, noncompetitive inhibitors decrease the
Vmax of the reaction.
2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of
substrate to enzyme. Thus, the enzyme shows the same Km in the presence or
absence of the noncompetitive inhibitor.
3. Effect on Lineweaver-Burke plot: Noncompetitive inhibition is readily
differentiated from competitive inhibition by plotting 1/v0 vs. 1/[S] and noting
that Vmax decreases in the presence of a noncompetitive inhibitor whereas Km
is unchanged.

Malonate is a Competitive inhibitor
Competitive inhibitor Increase Km and Vmax is same
Non Competitive inhibitor AfraTafreeh.com

decrease Vmax and Km is same
Diverging line on lineweaver burk plot Non Competitive inhibitor
Crossing line on lineweaver burk plot Competitive inhibitor
Parallel line on lineweaver burk plot Uncompetitive inhibitor

112 | Biochemistry
Worksheet
 AfraTafreeh.com

Enzyme | 113
Identify the type of inhibitor in the given plot
1
Vmax
1 1
Km Km
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114 | Biochemistry
Notes:
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9 Heme Metabolism
CONCEPTS
Â Concept 9.1: Heme synthesis, porphyria and
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heme degradation
116 | Biochemistry
Concept 9.1: Heme synthesis, porphyria and heme degradation
a) Enumerate various steps of heme biosynthesis
b) Define porphyria and enumerate various porphyria with their respective enzyme
deficiency
c) Describe the steps involved in heme degradation
Time Needed
1 Reading
st
100 mins
2 Reading
nd
30 mins
Concept Summary:
The complex heme molecule is synthesized from two simple precursor, glycine and succinyl
coenzyme A
It is partially mitochondrial and partially cytosolic process.
Heme biosynthesis occurs in most mammalian cells with the exception of mature erythrocytes
which do not contain mitochondria.
Approximately 85% of heme biosynthesis occurs in erythroid precursor cells of bone marrow
and majority of the remainder in hepatocytes.
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The steps of heme synthesis and various steps involved in porphyria are shown
in the figure below:
Heme Metabolism | 117
Porphyria
Group of disorders due to abnormalities in the pathway of biosynthesis of heme.
These can be genetic or acquired.
Porphyries can be classified as:
• Erythropoietic.
• Hepatic, on the basis of the organs or cells that are most affected.
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Porphyrins are cyclic compounds formed by the linkage of four pyrrole rings through
methyl (-HC=O) bridges.
Enzyme blocks later in the pathway result in accumulation of porphyrinogens and their
corresponding oxidation products i.e. porphyrins.
Porphyrins, when exposed to light at about 405 nm, are thought to become excited and
then react with molecular oxygen to form oxygen radicals, which then injure lysosomes
degradative enzymes, causing variable degrees of skin damage including scarring.
Porphyrins in the urine gives Soret band which shows the peak absorbance of light at
405 nm.
118 | Biochemistry
Soret band
Heme degradation
Heme is degraded in a multistep process and is finally converted to bilirubin which being
a toxic compound is excreted from the body after conjugation in the liver.
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Steps involved in heme degradation are enumerated below:
1 Conversion of heme to biliverdin is catalysed by heme oxygenase.

2 Biliverdin is reduced by (NADPH) to bilirubin, which is transported to the liver bound
to serum albumin.
3 In the liver, bilirubin is conjugated with glucuronic acid by enzyme Uridine
diphosphate (UDP)-glucuronyl transferase. The bilirubin diglucuronide that is formed
is soluble and is secreted into the bile.
4 Bilirubin diglucuronide is hydrolysed to free bilirubin in the bowel, their it is
converted urobilinogens and stercobilinogen, which are excreted in the urine and
faeces.

Rate limiting step of heme biosynthesis ALA synthase I
Major organ for synthesis of heme Bone marrow followed by liver
Soret band is seen in urine Because of porphyrin absorbing light at 405 nm
Lead poisoning affects two enzymes in heme ALA dehydratase and ferro chelatase
biosynthesis.
Ferro chelatase enzyme Incorporates iron within the porphyrinogen IX ring
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120 | Biochemistry
Worksheet
 AfraTafreeh.com

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122 | Biochemistry
Notes:
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10 Electron Transport Chain
CONCEPTS
Â Concept 10.1: Arrangement of Five Complexes
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of ETC
Â Concept 10.2: Chemio-osmotic Model
Â Concept 10.3: Factors Affecting the Oxidative
Phosphorylation
124 | Biochemistry
Concept 10.1: ETC arrangement
a) Describe arrangement of various enzyme complexes in ETC
b) Discuss chemiosmotic model for oxidative phosphorylation
Time Needed
1 Reading
st
60 mins
2 Reading
nd
30 mins
CONCEPT SUMMARY:
Location: The enzymes of electron transport chain are located in inner mitochondrial
membrane
1. Complex I is point of entry into ETC electrons from NADH
Key points related to this complex are enumerated below:
i. This enzyme complex is called NADH-coenzyme Q Reductase or NADH
dehydrogenase.
ii. Prosthetic groups is FMN, Fe-S.
iii. The electron acceptor from complex I is coenzyme Q.
iv. Inhibitors affecting the complex I are Rotenone, Amobarbital, piericidin
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2. Complex II is the point of entry into the electron transport chain for electrons
from succinate.
A. This enzyme complex is called Succinate- Coenzyme Q-reductase.
Coenzyme Q highly lipid soluble molecule firmly embedded in membrane. It
accepts electrons from both complex I and complex II and donates electrons
to complex III.
3. Complex III is electron acceptor for coenzyme Q:
A. This enzyme complex is called cytochrome c reductase.
B. Prosthetic groups are cytochrome b, c1,c and Fe-S.
C. Electron acceptor from complex III is cytochrome c.
D. Inhibitors blocking complex III are BAL and antimycin A.
Cytochrome c: Cytochrome c mediates transfer of electron from complex III to
complex IV. It is the only mobile cytochrome. Prosthetic group is Heme.
4. Complex IV is the electron acceptor for cytochrome c.
The components of the respiratory chain are arranged in order of increasing redox
potential.
Electron Transport Chain | 125
Figure Facts
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Figure: Arrangement of various complexes of electron transport chain

126 | Biochemistry
Concept 10.2: Chemiosmotic Model of Oxidative Phosphorylation
Time Needed
1st Reading 60 mins
2 Reading
nd
30 mins
Concept Summary:
Oxidative Phosphorylation:
Is the process is which ATP is formed as a result of transfer of electrons from NADH
or FADH2 to O2 by a series of electron carriers.
The flow of electrons from NADH or FADH2 to O2 through protein complexes located
in inner mitochondrial membrane leads to pumping of protons out of mitochondrial
matrix generating a proton motive force.
Chemiosmotic Theory of Oxidative Phosphorylation:

1. An electrochemical gradient of protons H+ across the mitochondrial inner
membrane, serves to couple energy flow of electron transport to the formation of
ATP.
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2. In electron transport chain, as electron moves down the chain, H+ is transferred
from mitochondrial matrix to the intermembrane space.
3. The protons in the intermembrane space pass through inner membrane and back
into matrix by ATP synthase.
4. ATP synthase is the complex that synthesizes ATP.
ATP Synthase Consists of Two Units:

• F0- spans the membrane and is composed of four subunits.
• It is the channel through protons cross the membrane.
• F1 units is composed of five subunits and contains catalytic site for ATP synthesis.
Inhibitors of ATP synthase – Oligomycin:

Binds with ATP synthase and blocks the proton channel.
Uncouplers of Oxidative phosphorylation.
• The action of uncouplers is to dissociate oxidation in the respiratory chain from
phosphorylation.
• This results in respiration becoming uncontrolled, since the concentration of ADP
or P; no longer limits the rate of respiration.
• Examples are – 2,4 Dinitrophenol, Dinitrocresol, pentachlorophenol and
Chlorocarbonyl Cyano phenylhydrazone.
• Physiological uncouplers are thyroxine, long chain fatty acids.
Point of entry into ETC electrons from NADH Complex I
Complex I is also called as NADH-coenzyme Q oxidoreductase
Inhibitors affecting the complex I are Rotenone, Amobarbital, piericidin
Complex II is also known as Succinate- Coenzyme Q-reductase.
Complex III Prosthetic groups are cytochrome b, c1,c and Fe-S.
Uncouplers of Oxidative phosphorylation 2,4 Dinitrophenol, Dinitrocresol, pentachlorophenol
and Chlorocarbonyl Cyano phenylhydrazone.
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128 | Biochemistry
Worksheet
 AfraTafreeh.com

Label the number of protons moving to intermembranous space at each compex
ELECTRON TRANSPORT CHAIN
AfraTafreeh.com
130 | Biochemistry
Notes:
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11 Genetics
CONCEPTS
Â Concept 11.1: Nucleotide Chemistry and
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Polynucleotide
Â Concept 11.2: Metabolism of Nucleotides
132 | Biochemistry
Concept 11.1: Nucleotide Chemistry and Polynucleotide
a) Describe the components of a nucleotide
b) Describe DNA and RNA structure and mention various types of RNA
Time Needed
1 Reading
st
60 mins
2 Reading
nd
30 mins
Concept Summary:
Nucleotide structure:
Following are the components of a nucleotide
1) Pentose sugar: it may be
a. Ribose sugar.
b. Deoxyribose sugar
2) Base: A nitrogenous base is attached by a glycosidic bond to the 1’ carbon atom
of the nucleotide’s sugar. Base may be:
a. Purines: adenine (A) and guanine (G) or
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b. Pyrimidines: Cytosine (C), thymine (T), and uracil (U).
3) Phosphate
Structure of a typical nucleotide
Polynucleotides
DNA and RNA are polynucleotides.
Nucleoside monophosphate are attached to each other via phosphodiester bond to
make a polynucleotide. Phosphodiester bonds is formed between the 3’ hydroxyl
of the pentose of one nucleotide and 5’ phosphate of another nucleotide.
Genetics | 133
DNA:
Key points regarding the structure of DNA are
• It contains deoxyribose sugar moiety.
• Double stranded.
• Each strand possesses polarity and they are antiparallel.
• The two strands of double stranded helix are held by hydrogen bonds between
the purines and pyrimidine bases of the respective linear molecule. Adenine pairs
with thymine with two hydrogen bonds.
• In the double stranded DNA molecule, the genetic information resides in the
sequence of nucleotides on one strand, the template strand. (also known as
NONCODING STRAND).
• The opposite strand is considered the CODING STRAND because it matches the
RNA transcript that encodes the protein.
RNA:
Key points regarding the structure of RNA are
• In RNA, the sugar moiety is a ribose.
• Single stranded molecule.
• It contains uracil as a base in place of thymine of DNA.
• AfraTafreeh.com
Its ‘A’ content is not equal to “C’ content.
• The sequence of the RNA molecule (except for U replacing T) is the same as that
of the coding strand of the gene.
Histones:
Key points regarding the histone protein
• These are basic proteins binding to DNA.
• These are of following types: H1, H2A,H2B, H3, H4.
• H2A and H2B are lysine rich and form dimers.
• H3 and H4 are arginine rich and forms tetramer.
134 | Biochemistry
Types of RNA:
1. Messenger RNA (mRNA).
2. Ribosomal RNA (rRNA).
3. Transfer RNA (tRNA).
4. Small nuclear RNA (snRNA).
5. Heteronuclear RNA (hn RNA).
Transfer RNA
• All tRNAs contain 4 main arms. The acceptor arm consists of a base paired stem
that terminates in the sequence C-C-A (5’ to 3’). It is through an ester bond to
the 3’-hydroxyl group of the adenosyl moiety that the carboxyl groups of amino
acids are attached.
• The anticodon arm recognizes the triplet nucleotide or codon of the template
mRNA. It has a nucleotide sequence complementary to the codon and is responsible
for the specificity of the tRNA.
• D arm is named for the presence of the base dihydrouridine. It is important for
proper recognition of a given tRNA by its proper amino acyl tRNA synthetase.
• TUC arm is named for the sequence, thymidine, pseudouridine it is involved in binding
of the amino acyl tRNA to the ribosomal surface at the site of protein synthesis.
• The extra arm is most variable arm in the tRNA
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Figure facts
Genetics | 135
Phosphodiester bonds formed as 3’ hydroxyl of the pentose of one nucleotide and 5’
phosphate of another nucleotide
Adenine pairs with thymine With two hydrogen bonds
H2A and H2B are lysine rich and Form dimers
H3 and H4 are arginine rich and Forms tetramer
D arm function is Amino acyl tRNA synthetase selection
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136 | Biochemistry
Worksheet
 AfraTafreeh.com

Genetics | 137
Enumerate various arms of tRNA and their role
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Notes:
138 | Biochemistry
Concept 11.2: Metabolism of Nucleotides
a) Describe steps of purine nucleotide
b) Describe steps of pyrimidine nucleotide
c) Discuss catabolism of purine nucleotide with associated disorder
Time Needed
1 Reading
st
60 mins
2 Reading
nd
30 mins
Concept Summary:
Biosynthesis of purine nucleotides:
Three processes contribute to biosynthesis of purine nucleotides:
1 Synthesis from amphibolic inter-mediates.
2 Phosphoribosylation of purines.
3 Phosphorylation of purine nucleosides.
Synthesis from Amphibolic Intermediates:

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• Inosine monophosphate is parent nucleotide from which AMP and GMP are formed.
• Synthesis of IMP from amphibolic intermediates Ribose -5- Phosphate involves
linear sequence of 11 reactions.
Contributors of various atoms in purine ring are as follows
The steps of purine nucleotide are shown below
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Genetics
|
Purine biosynthesis from ribose 5-phosphate and ATP

139
140
|
Biochemistry
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Conversion of IMP to AMP and GMP

Genetics | 141
Biosynthesis of pyrimidine nucleotides:
It is purely cytosolic process and the precursor molecule are CO2, Glutamine.
Steps are shown below:
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142 | Biochemistry
Catabolism of Purine nucleotide

Humans convert major purine nucleosides adenosine and guanosine to uric acid.
Metabolic disorders of Purine Catabolism:

1. Gout: In hyperuricemia, serum urate levels exceed the solubility limit, resulting
in crystallization of sodium urate in soft tissues and joints forms deposits called
tophi, causing an inflammatory reaction, acute gouty arthritis, which can progress
to chronic gouty arthritis. Visualization under a polarizing light microscope of
needle–shaped, intensively negatively birefringent crystals of sodium urate in
joint fluid is diagnostic of gout. The crystals appear yellow when their long axis
is parallel to the plane of polarized light and blue when perpendicular to
it.
2. Lesch Nyhan Syndrome:
X-linked recessive disorder.
Complete deficiency of enzyme HGPRTase, an enzyme of purine salvage,
results in purine overproduction.
3. This disease is characterized by hyperuricemia, uric acid lithiasis and a
syndrome of self mutilation.
4. Adenosine deaminase deficiency is associated with severe combined
immunodeficiency disease in which both T and B cells are sparse dysfunctional.
5. Purine nucleoside phosphorylase deficiency is associated with a severe
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thymus derived Lymphocyte deficiency with apparently normal B cell function.
Catabolism of Pyrimidines:
• The end products of Pyrimidine catabolism are CO2, NH3, β-alanine and β-amino-
iso-butyrate.
• Over production is rarely associated with clinically significant abnormalities.

Lesch Nyhan Syndrome Complete deficiency of enzyme HGPRTase
Adenosine deaminase deficiency severe combined immunodeficiency disease in
which both T and B cells are sparse
Inosine monophosphate is parent nucleotide from which AMP and GMP are formed
end products of Pyrimidine catabolism are CO2, NH3, β-alanine and β-aminoisobuytyrate
Genetics | 143
Worksheet
 AfraTafreeh.com

144 | Biochemistry
Discuss key steps of purine nucleotide biosynthesis
Discuss key steps of pyrimidine nucleotide biosynthesis
AfraTafreeh.com
How uric acid is produced from purine nucleotide degradation
Notes:
12 Elementary Genetics
CONCEPTS
Â Concept 12.1: DNA Replication
Â Concept 12.2: Transcription
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Â Concept 12.3: Translation
146 | Biochemistry
Concept 12.1: DNA Replication
a) Describe DNA synthesis
b) Discuss various mechanism of DNA repair
Time Needed
1 Reading
st
75 mins
2 Reading
nd
25 mins
Concept Summary:
DNA Replication:
Replication of DNA occurs only at a specified time during cell cycle. Known
as synthetic phase or “S phase”. Replication of DNA is semiconservative
process where new set of DNA has one parent strand and one daughter
strand.
On leading strand, DNA is synthesized continuously (forward strand).
On lagging strand, DNA is synthesized in short fragments called as OKAZAKI
fragments.
After many okazaki fragments are generated, the replication complex, begins to
remove the RNA primers, to fill in the gaps and to seal the fragments by enzymes
known as DNA ligases.
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In mammalian nuclear genome, RNA primers are eventually removed as part of
replication process
Different DNA polymerases share three important properties:
a. Chain elongation.
b. Processivity.
c. Proofreading.
1. Chain elongation: Rate at which polymerization occurs (no. of nucleotides/ sec).
2. Processivity: No. of nucleotides added to the nascent chain before the polymerase
disengages from the template.
3. Proofreading: Identifies copying errors and corrects them.
DNA topoisomerases: Causes unwinding of DNA.
DNA gyrase: It is bacterial topoisomerase (type 2 in E. coli) inhibited by nalidixic
acid and novobiocin.
DNA Repair:
Damage to DNA by environmental, physical and chemical agents is of 4
types:
a. Single base alteration.
b. Two base alteration.
c. Chain breaks.
d. Cross linkage.
Elementary Genetics | 147
Mechanism of DNA repair:

1. Mismatch repair.
2. Base-excision repair.
3. Nucleotide excision repair.
4. Double strand break repair.
Mechanism Problem Solution

Mismatch repair Copying errors (single base or two-to Methyl-directed strand cutting,
five – base unpaired loops) exonuclease digestion, and replacement
Base excision-repair Spontaneous, chemical, or radiation Base removal by N-glycosylase, abasic
damage to a single base sugar removal, replacement
Nucleotide excision Spontaneous, chemical, or radiation Removal of an approximately
repair damage to a DNA segment 30-nucleotide oligomer and replacement
Double- strand Ionizing radiation, chemotherapy, Synapsis, unwinding, alignment ligation
break repair oxidative free radicals
Figure facts
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148 | Biochemistry
Concept 12.2: Transcription
a) Describe transcription
b) Discuss various RNA polymerases
Time Needed
1 Reading
st
40 mins
2 Reading
nd
20 mins
Concept Summary:
Transcription (RNA Synthesis):
Synthesis of RNA from the gene is known as transcription
• Involves initiation, elongation and termination with 5’ to 3’ polarity.
• Ribonucleotides are used.
• Only a very small portion of genome is transcribed at one time.
• No primer is involved.
• No proofreading during transcription.
Transcription occurs in three phases
• Initiation
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• Elongation
• Termination
Key Points
• RNA polymerases in prokaryote is of one subtype
• RNA polymerase in eukaryote is of three subtype[ I,II,III]
• RNA polymerase has only 5-3 polymerase activity nd has no 3-5 exonuclease
activity. RNA synthesis thus occurs without proof reading.
• Termination of transcription in prokaryote may be rho dependent or rho
independent.
Fig: Process of transcription

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150 | Biochemistry
Concept 12.3: Translation
a) Describe translation
b) Describe mechanism of action od various inhibitors of translation
Time Needed
1 Reading
st
45 mins
2 Reading
nd
20 mins
Concept Summary:
Translation:
mRNA is translated in 5’ to 3’ direction. A protein is synthesized in the amino to
carboxyl direction.
Elongation factors are responsible for translocation of ribosome on mRNA WHICH
HELPS IN ELONGATION of protein synthesis.
Peptidyl transferase is the ribozyme which is responsible for making of peptide bond.
Four high energy equivalent is used for making of one peptide bond.
Termination is carried out by a single release factor eRF, a GTP driven protein. After
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multiple cycles of elongation, the nonsense codon of mRNA appears in the A site,
which is recognized by release factor This factor, in conjunction with GTP and the
peptidyl transferase promotes the hydrolysis of the bond between the peptide and
the tRNA occupying P site.
Fig.: Steps of protein synthesis

Polysomes:
These are assemblies of ribosomes on mRNA. In other words multiple ribosomes on the
same mRNA molecule form a polyribosome.
Following table explain the mechanism of action of certain drugs which inhibit
protein synthesis.
Inhibitors of Protein Synthesis:

Tetracycline Prevents the binding of aminoacyl tRNA to A site
Chloromycetin and macrolide class of antibiotics Bind to 23S rRNA, which has a role in peptide bond
formation
Puromycin AfraTafreeh.com
Structural analog of tyrosinyl-tRNA
Cycloheximide Inhibits peptidyl transferase in 70s ribosomal subunit
Diphtheria toxin Catalyses ADP- ribosylation of eEF-2 in mammalian

cells and inactivates it.

Direction of DNA synthesis 5-3 direction
Proof reading of DNA 3-5 exonuclease activity
Xeroderma pigmentosa is due to Defective nucleotide excision repair
mRNA is synthesized from RNA POLYMERASE II
rRNA is synthesized from RNA polymerase I
Codons on mRNA are read in 5-3 direction
Matching of codon and anticodon are Antiparallel
One peptide bond synthesis require 4 high energy bond

152 | Biochemistry
Worksheet
 AfraTafreeh.com

Mention steps of DNA synthesis.
What is the role of diphtheria toxin ?
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154 | Biochemistry
Notes:
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13 Genetic Technologies
CONCEPTS
Â Concept 13.1: Genetic Technologies
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156 | Biochemistry
Concept 13.1: Genetic technologies
a) Describe various technologies which are using basics of genetic principles
b) Discuss their clinical applcation
Time Needed
1 Reading
st
105 mins
2 Reading
nd
45 mins
Concept Summary:
Definition of Key Terminologies
Recombinant DNA Technology:
Isolation and manipulation of DNA, including end-to-end joining of sequences from
very different sources to make chimeric molecules (eg. Molecules containing both
human and bacteria DNA sequences in a sequence independent fashion), is the
essence of recombinant DNA research.
These novel combinations can be cloned, amplified manifold, by introducing them
into suitable cells, where they are replicated by the DNA synthesizing machinery of
the host. AfraTafreeh.com
Restriction Enzymes:
• These are endonucleases, enzymes that cut DNA at specific DNA sequences.
• Each enzyme recognizes and cleaves a specific double-stranded DNA sequence
(4-7 bp long).
• DNA cuts result in blunt ends or overlapping (sticky) ends. Sticky ends are
particularly useful in constructing hybrid or chimeric DNA molecules.
• A restriction map can be constructed for a given piece of DNA, that has a
characteristic linear array of sites for the various enzymes.
Genetic Technologies | 157
Restriction endonuclease enzyme
Vectors of Choice:
1. Plasmids.
2. Bacteriophages
3. Cosmids.
Plasmids are naturally occurring circular duplex DNA molecules ranging in size
from 2 kb to several hundred kbs. These exist as single or multiple copies within
the bacterium and replicate independently from the bacterial DNA. Plasmids accept
sequences 6-10 kb long. AfraTafreeh.com
Bacteriophages: Multiply within a host and lyse it (lytic pathway), or its DNA can
become integrated into host genome (lysogenic pathway).
Phages can accept DNA fragments 10-20 kbs long.
Cosmids combine the best features of plasmids and phages. Cosmids are plasmids
that contain the DNA sequences required for packaging lambda DNA into the phage
particle. Cosmids can carry inserts of chimeric DNA that are 35-50 kb long.
Even larger pieces of DNA (several hundred kilobases) can be incorporated into
• Bacterial artificial chromosome (BAC).
• Yeast artificial chromosome (YAC).
• P1 vectors.
These have largely replaced the plasmids, cosmids and phage vectors.
Cloning:
A clone is a large population of identical molecules, bacteria or cells, that arise from
a common ancestor. Cloning allows for the production of a large number of identical
DNA molecules.
This technique is based on the fact that chimeric or hybrid DNA molecules can be
constructed in cloning vectors, typically bacterial plasmids, phages or cosmids, which
then continue to replicate in a host cell under their own control systems. In this way,
the chimeric DNA is amplified.
158 | Biochemistry
Libraries:
The combination of restriction enzymes and various cloning vectors allows the entire
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genome of an organism to be packed into a vector. A collection of these different
recombinant clones is called a library.
Genomic Library: It is prepared from the total DNA of a cell line or tissue.
cDNA library: It represents the population of mRNAs in a tissue.
Expression Vector:
A vector in which the protein coded by the
Probes:
a. These are generally pieces of DNA or RNA labeled with 32P – containing nucleo tide
and these recognize a complementary sequence to be effective.
cDNA probes are used to detect DNA fragments on southern blot transfers and to
detect and quantitate RNA on northern blot transfers.
b. Specific antibodies can also be used as probes, provided that the vector used
synthesizes protein molecules that are recognized by them.
The Polymerase Chain Reaction:

It is a method of amplifying a target sequence of DNA. It is a sensitive, selective and
extremely rapid means of amplifying a DNA sequences as short as 50-100 bp and
as long as 10kb
Twenty cycles provide an amplification of 106 and 30 cycles of 109.
It allows the DNA in a single cell, hair follicle or sperm to be amplified and analyzed.
Application of PCR:
1. In Forensic medicine.
2. To detect infectious agents, especially latent viruses.
3. To make prenatal genetic diagnoses.
4. To detect allelic polymorphisms.
5. To establish precise tissue types for transplants.
6. To study evolution, using DNA from archeological samples.
Repeated cycles of heat denaturation, annealing of the primers to their complementary
sequences and extension of the annealed primers with DNA polymerase result in an
exponential amplification of DNA segments of defined length.
E.coli DNA polymerase used in early PCR reactions was destroyed by each heat
denaturation cycle. Substitution of a heat stable DNA polymerase from ‘Thermus
aquaticus’ an organism that lives and replicates at 700 - 800 C, obviates this problem
and has allowed for automation of the reaction.
Various Novel Techniques of PCR:

Reverse Transcription Polymerase Chain Reaction:
In, molecular biology reverse transcription polymerase chain reaction (RT-PCR) is
a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA)
molecule. The RNA strand is first reverse transcribed into its DNA complement or
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complementary DNA, followed by amplification of the resulting DNA using polymerase
chain reaction.
Presently the COVID 19 virus RNA is amplified using RT-PCR technique
Reverse transcription PCR is not to be confused with real-time polymerase chain
reaction which is also marketed as RT-PCR.
Processes:
In the first step of RT-PCR, called the “first strand reaction,” complementary DNA
is made from a messenger RNA template using dNTPs and an RNA-dependent DNA
polymerase, reverse transcriptase, through the process of reverse transcription. The
above components are combined with a DNA primer in a reverse transcriptase
buffer for an hour at 37°C.
After the reverse transcriptase reaction is complete, and complementary DNA has
been generated from the original single- stranded mRNA, standard polymerase chain
reaction, termed the “second strand reaction,” is initiated.
1. A thermostable DNA polymerase and the upstream and downstream DNA primers
are added.
2. The reaction is heated to temperatures above 37°C to facilitate sequence specific
binding of DNA primers to the cDNA
3. Further heating allow the thermostable DNA polymerase (‘Transcriptase’) to make
double-stranded DNA from the primer bound cDNA.
4. The reaction is heated to approximately 95°C to separate the two DNA strands
5. The reaction is cooled enabling the primers to bind again and the cycle repeats.
160 | Biochemistry
After approximately 30 cycles, millions of copies of the sequence of interest are

generated. The original RNA template is degraded by RNase H, leaving pure cDNA
(plus spare primers).
Usage of Reverse Transcription Polymerase Chain Reaction:
The exponential amplification via reverse transcription polymerase chain reaction
provides for a highly sensitive technique, where very low copy number of RNA
molecules can be detected. Reverse transcription polymerase chain reaction is widely
used in the diagnosis of genetic diseases and, quantitatively, in the determination of
the abundance of specific different RNA molecules within a cell or tissue as a measure
of gene expression. Northern blot is used to study the RNA’s gene expression further.
Quantitative Polymerase Chain Reaction:

Quantitative polymerase chain reaction (Q-PCR) is a modification of polymerase
chain reaction used to rapidly measure the quantity of a product of polymerase chain
reaction. It is preferably done in real-time, thus is an indirect method for quantitatively
measuring starting amounts of DNA, complementary DNA or ribonucleic acid (RNA). It is
commonly used to determine whether a genetic sequence is present, and if it is present,
to determine the number of copies in the sample. There are 3 methods which vary in
difficulty and detail. Like other forms of polymerase chain reaction, the process is used
to amplify DNA samples, via the temperature-mediated enzyme DNA polymerase.
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The three commonly used methods of quantitative polymerase chain reaction are
through agarose gel electrophoresis, the use of SYBR Green, a double stranded DNA
dye, and the fluroscent reporter probe. The latter two of these three can be analysed in
real-time, forming real-time polymerase chain reaction.
Although real-time quantitative polymerase chain reaction is often marketed as RT- PCR,
it should not to be confused with reverse transcription polymerase chain reaction, also
known as RT-PCR.
Real-time Polymerase Chain Reaction:

In molecular biology, real-time polymerase chain reaction, also called quantitative
real time polymerase chain reaction (QRT-PCR) or kinetic polymerase chain
reaction, is a laboratory technique used to simultaneously quantify and amplify a
specific part of a given DNA molecule. It is used to determine whether or not a specific
sequence is present in the sample; and if it is present, the number of copies in the
sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR),
itself a modification of polymerase chain reaction.
The procedure follows the general pattern of polymerase chain reaction, but the DNA
is quantified after each round of amplification; this is the “real-time” aspect of it. Two
common methods of quantification are the use of fluorescent dyes that intercalate
with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when
hybridized with a complementary DNA.
Frequently, real-time polymerase chain reaction is combined with reverse transcription
polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling
a researcher to quantify relative gene expression at a particular time, or in a particular
cell or tissue type.
Although real-time quantitative polymerase chain reaction is often marketed as RT- PCR,
it should not to be confused with reverse transcription polymerase chain reaction, also
known as RT-PCR.
Applications of real-time Polymerase Chain Reaction:

There are numerous potential applications for the technique of real-time polymerase
chain reaction in the laboratory. Often an investigator will want to know how the
genetic expression of a particular gene changes over time, such as during germination,
or in response to changes in environmental conditions. Real-time polymerase chain
reaction has been used to detect changes in gene expression in a tissue in response
to an administered pharmacological agent and is thus an important technique in drug
discovery and testing. In recent years, real-time polymerase chain reaction has been
slightly superseded by DNA microarray technology, which allows the expression of many
genes to be quantified in a cell sample instead of just one. However, a standard
real-time polymerase chain reaction experiment is still cheaper and easier to set up
than an average microarray, and so remains an important tool in molecular biology labs.
Figure facts
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Polymerase chain reaction

Southern blotting To analyse DNA
Northern blotting To find out gene expression
Western blotting To study protein characteristic
Dideoxy nucleotide trail sequencing Sanger’s technique of DNA sequencing
RT PCR For amplification of RNA
Real time PCR Quantitative PCR
162 | Biochemistry
Worksheet
 AfraTafreeh.com

Enumerate various steps of PCR
Mention the names of real time dye
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164 | Biochemistry
Notes:
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14 Micronutrients
(Vitamins and Mineral)
CONCEPTS
Â Concept 14.1: Fat Soluble Vitamin
Â Concept 14.2: Water Soluble Vitamin
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166 | Biochemistry
Concept 14.1: Fat Soluble Vitamin
a) Classify vitamins
b) Enumerate various aspect of fat soluble vitamins
Time Needed
1 Reading
st
100 mins
2 Reading
nd
45 mins
Concept Summary:
Vitamins are broadly classified into water
soluble and fat soluble types. Fat soluble – Vitamin A, D, E, K.
Water soluble – Vitamin B complex , vitamin C.
Fat Soluble Vitamin

Vitamin A:
All compounds with Vit. A activity are referred to as Retinoids.
Active forms are:
• Retinol. AfraTafreeh.com
• Retinal.
• Retinoic Acid.
β-Carotene:
It is Provitamin A.
2 retinals are joined by polyisoprenoid chain.
It has 1/6th as effective as vitamin A.
Digestion, absorption and transport of vitamin A:
A. Retinol esters present in the diet are hydrolyzed in the intestinal mucosa,
releasing retinal and free fatty acids.
B. Retinol derived from esters and from the cleavage and reduction of carotenes is
reesterified to long-chain fatty acids in the intestinal mucosa and secreted as a
component of chylomicrons into the lymphatic system.
C. Retinol esters contained in chylomicrons are taken up by and stored in the liver
in the form of retinal palmitate
D. When needed, retinal is released from the liver and transported to extrahepatic
tissues by the plasma retinal-binding protein (RBP).
E. The retinol-RBP complex attaches to specific receptors on the surface of the cells
of peripheral tissues, permitting retinol to enter.
F. Many tissues contain a cellular retinol-binding protein that carrier retinol to sites
in the nucleus where the vitamin acts in a manner analogous to steroid hormones.
Micronutrients (Vitamins and Mineral) | 167
Functions of vitamin A:
• Retinal: vision.
• Retinoic acid: growth and differentiation.
• Retinol: reproductive system.
Distribution of vitamin A:
Liver, kidney, cream, butter, and egg yolk are good sources of preformed vitamin
A. Yellow and dark green vegetables and fruits are good dietary sources of the carotenes,
which serve as precursors of vitamin A.
Requirement for vitamin A:
• The RDA for adults is 1000 retinol equivalents (RE) for males and 800 RE for females.
• One RE = 1 mg of retinol / 6 mg of beta carotene / 12 mg of other carotenoids.
Deficiency:
Night blindness, -Bitot’s spots, Keratomalacia.
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Follicular hyperkeratosis, Gonadal dysfunction.

168 | Biochemistry
Toxicity of Retinoids:
Vitamin A: Excessive intake of vitamin A produces a toxic syndrome called
hypervitaminosis A.
Amounts exceeding 7.5 mg/day of retinol should be avoided.
Early signs of chronic hypervitaminosis A are reflected in the skin, which becomes
dry and pruritic, the liver, which becomes enlarged and can become cirrhotic, and in
the nervous system, where a rise in intracranial pressure may mimic the symptoms
of a brain tumor. Pregnant women particularly should not ingest excessive quantities
of vitamin a because of its potential for causing congenital malformations in the
developing fetus.
Vitamin D:
Vitamin D is a steroid prohormone.
Source of vitamin D.
a. Diet: Ergocalciferol (Vitamin D2) – from plants. Cholecalciferol (Vitamin D3 ) –
from animal tissues.
b. Endogenous: 7 – Dehydrocholesterol is converted to cholecalciferol in the dermis
and epidermis of humans exposed to sunlight.
Metabolism of Vitamin D: AfraTafreeh.com
• Vit D2 and Vit D3 are not biologically active, but are converted in vivo to the active
form by two sequential hydroxylation reactions.
• The first hydroxylation takes place at the 25th-position in the liver catalyzed by
25-hydroxylase.
• The product 25-OH D3 is the predominant form of Vit D in the plasma and the
major storage form of the vitamin.
• 1-hydroxylase found in kidneys primarily, hydroxylates 25-OH D3 at the 1-position,
resulting in the formation of 1,25-dihydroxy cholecalciferol
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Cholecalciferol can be hydroxylated at the C25 position by a fiver enzyme. The 25 hydroxycholecalciferol further
metabolized to 1a,25-dihydroxycholecalciferol or to 24, 25 dihydroxycholecalciferol
• Both hydroxylases employ cytochrome P450, molecular oxygen and NADPH.

• 1,25-dihydroxy cholecalciferol is the most potent Vit D metabolite, its formation is
tightly regulated by the levels of plasma phosphate and calcium ions.
Function of Vit D:
Overall function of 1,25-dihydroxy cholecalciferol is to maintain adequate plasma levels
of calcium. It performs this function by
a. Increasing uptake of calcium by the intestine.
b. Minimizing loss of calcium by the kidneys.
c. Stimulating resorption of bone, when necessary.
Calcium uptake is enhanced by the increased synthesis of a specific calcium binding
protein, calbindin.
Distribution and requirement of Vit D:
• Occurs naturally fish, liver and egg yolk. Milk, unless artificially fortified, is not a good
source of Vitamin D.
170 | Biochemistry
The Vitamin D Receptor and mechanism of action
The vitamin D receptor forms a complex with another intracellular receptor, the retinoid-X
receptor, and this heterodimer binds to DNA.
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The vitamin D receptor binds several forms of cholecalciferol. Its affinity for 1,25-dihy-
droxy cholecalciferol is roughly 1000 times that for 25-hydroxycholecalciferol, which
explains their relative biological potencies.
Numerous effects of vitamin D on bone have been demonstrated. As a transcriptional
regulator of bone matrix proteins, it induces the expression of osteocalcin and suppresses
synthesis of type I collagen.
In cell cultures, vitamin D stimulates differentiation of osteoclasts.
Toxicity:
• Vitamin D is the most toxic vitamin.
• High doses (1,00,000 IU for weeks or months) can cause loss of appetite, nausea,
thirst and stupor.
• Enhanced calcium absorption and bone resorption results in hypercalcemia leading
to deposition of calcium in many organs (Chondrocalcinosis) particularly in kidneys
and arteries.
Vitamin E:
Consist of eight naturally occurring tocopherols, of which alpha tocopherol is the most
active.
• Primary function of vitamin E is as an antioxidant in prevention of non enzymatic
oxidation of cell components by molecular oxygen and free radicals.
Distribution and requirement of vitamin E:
• Vegetable oils are rich source of vitamin E, while liver and eggs contain moderate
amounts.
• RDA for alpha tocopherol is 10 mg for men and 8mg for women – vitamin E requirement
increases as the intake of PUFA increases.
Key points of vitamin E:
• Vitamin E is the most potent natural antioxidant.
• AfraTafreeh.com
It is a first line of defense against the free radicals.
• It is a chain terminating type of antioxidant.
• Acts synergistically with selenium.
Deficiency of vitamin E:
Almost entirely restricted to premature infants. Signs of human vitamin E deficiency
include sensitivity of RBC to peroxide, and the appearance of abnormal cellular
membrane.
Requirement:
• Men = 10 mg.
• Women= 8 mg.
• Pregnancy= 10 mg.
• Lactation =12 mg.
• More than 60 yrs =12 mg.
Toxicity:
• It is the least toxic of all fat-soluble vitamins.
• More than 1000 IU/day if taken for long time leads to toxicity.
Vitamin – K:
Vitamin K are Naphthoquinone derivatives with a long isoprenoid side chain.
Vitamin – K1 = Phylloquinone. Vitamin – K2 = Menaquinone. Vitamin – K3 = Menadione.
172 | Biochemistry
Factors dependent on vitamin K are II, VII, IX, and X. All these, factors are synthesized
in the liver as zymogens.
They undergo posttranslational modifications; gamma carboxylation of glutamic acid
residues. These are calcium ion binding sites. Prothrombin has 10 such residues. The
gamma carboxy glutamic acid (GCG) are also seen in CRP, bone osteocalcium, and
structural protein of lung, kidney and spleen.
Sources:
Plants, animals. Intestinal bacteria.
Causes of Deficiency:
• Fat malabsorption.
• Newborn. AfraTafreeh.com
• Antibiotic therapy.
Manifestation of Vitamin K Deficiency:
Hemorrhagic disease of new born. Prolong bleeding time, clotting time, prothrombin
time
Sources:
Plants, animals. Intestinal bacteria.
Causes of Deficiency:
• Fat malabsorption.
• Newborn.
• Antibiotic therapy.
Clinical feature of Deficiency:
Hemorrhagic disease of new born. Prolong BT, CT, PT.
Concept 14.2: Water Soluble Vitamin
a) Enumerate various aspect of water soluble vitamins
Time Needed
1 Reading
st
120 mins
2nd Reading 60 mins
Water Soluble Vitamins:

Thiamine (Vitamin B1):
Key points
• Made up of substituted Pyrimidine ring and substituted thiazole ring.
• Active form is thiamine diphosphate.
• Is heat labile.
• RDA = 1-1.5 mg/day.
Functions of TPP:
1. Oxidative decarboxylation (eg. PDH Complex).
2. Transketolation (eg. HMP Shunt pathway and formation of Ketones).
Dietary Sources: AfraTafreeh.com
Unrefined cereal grains.
Liver. Heart. Kidney.
Deficiency:
Causes:
• Ingestion of milled rice, raw fish, tea (has anti thiamine factor).
• Chronic alcoholic.
• Increased demand in increased muscle activity, prolonged fever, hypothyroidism.
Clinical Symptoms of deficiency:
Mental confusion, anorexia, muscle weakness, ataxia, ophthalmoplegia.
Muscle wasting, oedema (Wet type), tachycardia, enlarged heart, peripheral vasodilation,
biventricular myocardial failure, water and salt retention. peripheral neuropathy, amnesic
psychosis.
Assessment of Deficiency:
Estimation of deficiency is done by ratio of lactate and pyruvate and by erythrocyte trans
ketolase estimation.
Deficiency Syndromes:
• Wernicke’s encephalopathy.
• Korsakoff’s syndrome.
• Beriberi = dry / wet.
174 | Biochemistry
Riboflavin (Vitamin B2):
• Is photosensitive and heat stable, yellow
• fluorescent compound.
• Is 7, 8 dimethyl 10-isoalloxazine ring attached to sugar ribitol.
• Active form: FAD, FMN.
Sources:
Liver, kidney, heart. Vegetables.
Human and cow milk.
Requirement: 0.6 mg/1000 Cal of energy.
Functions:
Oxidation reduction reactions, one electron and 2 electron transfer reactions with
Sulphur containing compounds.
FMN and FAD are prosthetic groups of:
1. Dehydrogenases requiring FAD
Succinate dehydrogenase.
Alpha keto glutarate dehydrogenase.
Glycerol 3 phosphate dehydrogenase.
Acyl CoA dehydrogenase.
Dihydrolipoyl dehydrogenase.
2. Respiratory chain: AfraTafreeh.com
Complex 1 (NADH Dehydrogenase) (FMN).
Complex 2 (FAD).
3. Amino acid metabolism:
L-amino acid oxidase (FMN).
Xanthine oxidase.
4. Glutathione reductase (FAD).
Deficiency:
Sore throat, angular stomatitis, pharyngeal and oral mucosal oedema, magenta tongue,
(glossitis) seborrheic dermatitis and cheilosis.
Assessment done by: RBC glutathione reductase activity.
Niacin (Vit B3):

Not a true vitamin.
Coenzyme forms – NAD and NADP. Sources:- yeast, meat, liver, poultry, whole cereals
and legumes.
Rrequirement = 6.6 Niacin equivalents/1000 Cal of energy.
Functions: It functions as dehydrogenases in oxidoreductase reactions. Most of them
function reversibly.
NAD as Coenzyme:
1 Alcohol dehydrogenase (ethanol –→ acetaldehyde).
2 Lactate dehydrogenase (Pyruvic acid LA).
3 Malate dehydrogenase (Malate 0.AA).
4 Pyruvate dehydrogenase complex (P.A. Acetyl CoA).
5 〈-ketoglutarate dehydrogenase complex (〈-ketoglatarate – succinyl CoA).
NADP+ as coenzyme:
• Glucose – 6– Phosphosphate dehydrogenase (G–6–P, 6-Phosphogluconate).
• Glutathione reductase.
Either NAD+ or NADP+:
i. Glutamate dehydrogenase.
ii. Isocitrate dehydrogenase.
Deficiency leads to Pellagra:
Clinical symptoms are chronic wasting diseases and Dermatitis, Diarrhoea, Dementia,
and Death (4 D’s), is associated with achlorhydria, glossitis, stomatitis and vaginitis.
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Deficiency is due to:

• Diet (corn, wheat).
• Hartnup’s disease.
• Carcinoid syndrome.
• Shorghum.
• INH therapy.
Biotin (Vit B7):

Is cis tetrahydro – 2 oxo thienol 3,4 imidazoline derivative. Some Biotin is linked
noncovalently as a complex with avidin (protein in egg white).
Sources: Liver, kidney, egg, yeast, milk. Intestinal microflora make a significant
contribution to body pool.
Requirement = 100 – 200 ug.
176 | Biochemistry
Deficiency can be by sterilization of intestine and ingestion of raw egg. Dietary proteins
contain bound biocytin which is resistant to proteins by hydrolytic enzymes.
Active form is covalently bound to epsilon group of lysine residue of biotin dependent
apo-E.
Biotin is cleared from circulatory blood more rapidly in deficient than in normal people.
It is taken up by liver, muscle and kidneys and is localized in cytosolic and mitochondrial
carboxylases.
Deficiency: anorexia, nausea, vomiting, glossitis, pallor, depression, dry scaly dermatitis.
Diagnosis is by microbiological assay.
Pantothenic acid (Vitamin B5):

Is synthesized from pentoic acid. It is integral part of 4’ phosphopentathiene.
Which serves as a covalently attached acyl carrier protein and lies within the structure
of Coenzyme A.
Sources: egg yolk, kidney, liver, yeast, green vegetables and sweet potatoes.
It is heat labile and hygroscopic. Synthetic from = Calcium salt. Requirement = 4-7 mg/
day.
Deficiency: Postural hypotension. 1. Heart rate, epigastric distress, anorexia and
constipation. and burning feet syndrome.
Pyridoxine Vitamin (B6):

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Deficiency of this vitamin is especially seen in patient’s on INH (ATT). Has three natural
forms – Pyridoxine, Pyridoxamine, Pyridoxal.
These are 4 substituted 2 – Methyl 3 Hydroxyl 5-Hydroxy methylpyridines.
Coenzyme: PLP (pyridoxal – 5’ phosphate) Sources: Meat, poultry, fish, yeast, bran,
green leafy vegetables.
Estimation by:
1. Urinary excretion of vitamin B6.
2. Estimation of Xanthurenic acid after tryptophan load test.
3. Plasma levels of PLP.
4. RBC transaminase activity.
Functions: As coezyme in amino transferase reactions and forms Schiff’s base.
A wide range of reactions in which PLP acts as a cofactor are:
1. Transamination.
2. Decarboxylation.
3. Aldol cleavages.
4. Racemizations.
5. Deaminations.
6. PLP is also a cofactor of glycogen phosphorylase.
7. Coenzyme for kynureninase.
Deficiency – Convulsions, Dermatitis, Glossitis, Cheilosis.
Folic Acid:
Pteridine ring joined to para-aminobenzoic acid = pteroic acid and Glutamic acid.
Multiple forms of folate occur with substitution of functional groups as methyl, formyl,
methylene, hydroxymethyl, at various N-atoms in pteroic acid residues.
Function: Folate is essential for the transfer of single carbon units.
Major Reactions:
Serine –Glycine, Catabolism of histidine., synthesis of thymidylate, methionine, purines.
Reaction Group Transferred Folic Acid Derivative
Serine/Glycine metabolism Methylene (– CH2) N5N10 – Methylene THF N5
Histidine catabolism Thymidilate Formimino THF N5N10 Methylene
synthesis Methionine synthesis Formimino (CHNH) Methylene THF N5 MethylTHF
Purine synthesis – (CH2) Methyl (–CH3) Methenyl N5N10 Metheny1THF
(= C H–) N10 Formyl THF
Sources – yeast, liver, egg, green leafy vegetables, oil seeds.
RDA = 200ug/day.
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Vitamin – B12– Cobalamine or Corrinoids:

Have tetrapyrrole ring surrounding Cobalt atoms and nucleotide side chain attached to
Cobalt.
Cobalt – corrin – complex is called Cobamide.
Physiological forms are Methyl cobalamine and 5’ deoxyadenosyl cobalamine.
Absorption is with intrinsic factor.
Vitamin B12 is coenzyme for two physiologically important functions:
1 Synthesis of methionine.
2 Conversion of methylmalonic acid to succinic acid (requires methyl deoxyadenosyl
cobalamine).
Deficiency: Megaloblastic anaemia (Pernicious anaemia) and neuropathy.

178 | Biochemistry
Functions:
1. It acts as a cofactor for procollagen hydroxylases which hydroxylates prolyl and lysyl
residues, within nascent peptides in connective tissue, cartilage and dentin.
2. Hydroxylation of gamma butyro betaine to carnitine.
3. Tyrosine metabolism.
4. Microsomal drug metabolism.
5. Synthesis of epinephrine and anti-inflammatory steroids by adrenals.
Vitamin B12 deficiency can also lead to subacute degeneration of spinal cord.
Ascorbic Acid (Vitamin C):
Is enol form of 2-oxo-L-gulofuronolactone.
It is white crystalline substance readily soluble in water.
It is reversibly oxidized to dehydro ascorbate. Dehydro form is more labile. It is
destroyed by heat alkali and storage. Man cannot synthesize Vit. C because of absence
gulonolactone oxidase.
Sources:- Citrus fruits, berries, melons, tomatoes, green chillies and leafy vegetables.
Requirement: 60 mg/day.
Higher RDA of 100 mg is required for premature new born babies 30-35mg more is
required during lactation.
Deficiency–SCURVY: indicated by subcutaneous and other haemorrhages, muscle
weakness, soft swollen gums and loose teeth.
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Worksheet
 AfraTafreeh.com

180 | Biochemistry
Enumerate RDA of various water soluble vitamins
What all enzyme need FAD as cofactor
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Notes:
15 Nutrition and
Energy Metabolism
CONCEPTS
Â Concept 15.1: Energy Metabolism
Â Concept 15.1: Nutrition
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182 | Biochemistry
Concept 15.1: Energy Metabolism
a) Describe various factors affecting the BMR
b) Define RQ, SDA, Calorific value
c) Describe dietary fibers
Time Needed
1 Reading
st
60 mins
2 Reading
nd
30 mins
Concept Summary:
Caloric value of foods. Caloric value is defined as amount of heat energy obtained
by burning 1.0 gm. of food stuff completely in the presence of oxygen.
Caloric value of different food stuffs determined in a apparatus called bomb
calorimeter.
Dietary Sources of energy

Proteins — 4 kcal/g
Fats — 9 kcal/g
Carbohydrates — AfraTafreeh.com
4 kcal/g
BMR (Basal Metabolic Rate). The rate of energy production under basal condition
per unit time and per square meter of body surface is known as basal metabolic rate.
Basal conditions are:
1. Person should be awake but at complete physical and mental rest.
2. Person should be fasting.
3. Person should be in recumbent/reclining position in bed.
4. Person should be in normal condition of environment i.e., at normal tempera-
ture, pressure and humidity.
Factors influencing BMR:

1. Age. BMR of children is much higher than the adult.
2. Sex. Women normally have lower BMR than men.
3. Surface area. BMR is directly proportional to surface area expressed as C/Sqm/
hr.
4. State of nutrition. BMR lowered in condition of malnutrition starvation and
wasting diseases.
5. Drugs. Drugs like caffeine, Benzedrine, Epinephrine, Nicotine, Alcohol etc.
increase the BMR.
6. Hormones. Of thyroid, adrenal medulla and anterior pituitary increase BMR.
7. Pregnancy. BMR of pregnant mother after six months of gestation rises.
Nutrition | 183
Clinical Aspect (Pathological Variations in BMR):

1. Fever. Infections and febrile diseases elevate BMR usually in proportion to increase
in temperature.
2. Diseases. BMR increased in diseases characterized by increased activity of cells. e
g. Leukaemias and Polycythemia.
3. Endocrine diseases:
(a) BMR is increases in hyperthyrodism, Cushing disease, Cushing syndrome also
in Acromegaly.
(b) BMR is reduced in hypothyrodism and Addison disease.
Respiratory Quotient (RQ):

RQ is ratio of volume of CO2 produced by a volume of O2 consumed during a given
time. So, RQ is simply a ratio.
Normal RQ. In a healthy adult on a mixed diet, it is 0.85.
Factors affecting RQ:

Role of diet.
a. Carbohydrate – RQ is one.
C6H12O6 + 6O2 ⇒ 6 CO2 + 6H2O
CO2 produced 6 6
RO for carbohydrate = =
AfraTafreeh.com =1
O2 consumed 6 6
b. Fats – RQ is lowest and is about 0.7.

For e.g. Oxidation of tristearin.
C15H110O6 + 163O2 → 114CO2 + 110H2O
114
RQ for tristearin = = 0.7
163
c. Proteins - RQ for proteins has been calculated indirectly, is about 0.8
2C3H7O2N + 6O2 → (NH2).CO + 5CO2 + 5H2O
5
RQ = = 0.8
6
d. RQ of mixed diet – RQ is about 0.85.
Clinical Aspects:
1. In acidosis. During acidosis, CO2 output is greater than O2 consumption, hence
RQ is increases.
2. In alkalosis. RQ will fall, because respiration is depressed and CO2 retained in
body and less CO2 produced.
3. Febrile conditions. Increase RQ.
4. In diabetes mellitus. RQ will fall initially because energy is supplied by oxidation
of fats.
184 | Biochemistry
Specific Dynamic Action:

It is defined as an extra heat produced, over and above the actual heat ought to be
produced outside from a given amount of food when this food is metabolised inside
the body.
1. Proteins have greatest SDA, amounting to about 30% above its caloric value.
2. Carbohydrates cause an increase of about 5% or 6%.
3. Fats cause about 4%.
Energy requirement. The energy requirement of an individual is defined as level of
energy intake in relation to expenditure which is less likely to result in obesity and
heart disease.
Broadly, energy requirement of an individual is made of three components.
1. Energy required for basal metabolism (This is about 1 kcal/hr for every kg of body
weight for an adult)..
2. Energy required for daily activities such as, walking, sitting, standing, climbing
stairs etc.
3. Energy expenditure for occupational work, further classified as Light work,
Moderate work and Heavy work.
Balanced diet. A balanced diet is defined as one which contains a variety of foods
in such quantities and proportion that need for energy and all nutrients is adequately
met for maintaining health, vitality and general well being.
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Nutrition | 185
Concept 15.2: Nutrition
Time Needed
1st Reading 30 mins
2 Reading
nd
20 mins
Nutrients are organic and inorganic complexes contained in food.

1. Macronutrients:
Carbohydrates – 65-80%
Fats – 10-30%
Protein – 7-15%
2. Micronutrients are vitamins and minerals.
Fats:
Fats are classified as (1) Simple lipids, (2) Complex lipids, (3) Derived lipids.
• Most of body fat (99%) in adipose tissue is in form of triglycerides.
• Fatty acids are divided into (1) Saturated fatty acids such as lauric, polmitic
and stearic acid (2) Unsaturated fatty acids which are further divided into
monounsaturated (oleic acid) and polyunsaturated fatty acids.
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Sources:
1. Fats from plant sources. Contains unsaturated fatty acids except coconut oil and
palm oil consists primarily of saturated fatty acids, e. g. (Safflower oil, sunflower oil,
corn oil).
2. Fats from animal sources. Contains a higher proportion of saturated fatty acids
except fish oil.
Essential fatty acids. E.g. Linoleic acid, linolenic acid and arachidonic acid. The most
important essential fatty acid is Linoleic acid, which serves as a basis for production of
other essential fatty acids.
Functions:
1. Fats provide a concentrated source of energy about 9 kcal/gm.
2. Serve as vehicle for fat soluble vitamins.
3. Essential fatty acids are needed for body growth, the structural integrity of cell
membrane.
4. Diet rich in EFA reduce serum cholesterol and low density lipoprotein. Several
hypothesis have been proposed to explain it (i) Stimulation of cholesterol
excretion into intestine (ii) Stimulation of oxidation of cholesterol to bile
acids. (iii) Shift in distribution of cholesterol from plasma into the tissues because
of increased catabolic rate of LDL due to up regulation of LDL receptors.
5. PUFA are precursors of Prostaglandins.
186 | Biochemistry
Dietary goals. A reduction in fat consumption to 30% of total calories in recommended.
1. Saturated fat – 10%.
2. Monounsaturated fats – 10%.
3. Polyunsaturated fatty acids – 10%.
Dietary Fats and disease:
1. Obesity. It is expressed in terms of body mass index. A BMI of 30 or more in male
and 28.6 or more in female indicates obesity.
2. Phrenoderma. Deficiency of essential fatty acids in diet is associated with rough
and dry skin known as phrenoderma.
3. Coronary heart disease. LDL increases risk of coronary artery disease and HDL
exerts a protective effective against atherosclerosis.
4. Cancer. There has been some evidence that diet high in fat increases risk of colon
cancer and breast cancer.
Carbohydrates:
There are 3 main sources of carbohydrates viz. starches, sugar and cellulose.
1. Starch is abundant in cereals, roots and tubers.
2. Sugars comprise monosaccharides (glucose, fructose and galactose) and
disaccharides (sucrose, lactose and maltose). These sugars are easily assimilated.
3. Cellulose contributes to dietary fiber.
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Dietary fibers. Dietary fiber consist of nondigestible carbohydrates including cellulose,
lignin and pectin.
Beneficial effects of Dietary fibers:
1. Reduces constipation and hemorrhoid formation.
2. Increases bowel motility and reduces exposure of gut to carcinogens.
3. Lowers blood cholesterol.
4. Interferes with mineral absorption.
Dietary goal. Dietary goal is to increase carbohydrate intake from present 46% of total
calories to 58% from complex carbohydrates.
Nutrition | 187
Proteins:
Proteins are made up of amino acids.
Essential amino acids are: Leucine, Isoleucine Lysine, Methionine, Phenylalanine,
Threonine, Valine tryptophan.
Semiessential amino acids are: Arginine and Histidine.
A protein is biologically complete if it contains all the EAA in amounts corresponding
to human needs.
Supplementary action of Proteins:
Cereal proteins are deficient in Lysine and Threonine and pulse proteins in Methionine.
When two or more vegetarian foods are eaten together, these proteins supplement one
another and provide a protein comparable to animal protein in respect of EAA.
Evaluation of protein includes estimation of biological value, digestibility cofficient and
net proteins utilization. Net protein utilization is more practical because it is product of
biological and digestibility cofficient divided by 100.
Assessment of Protein Nutrition status:
1. Arm muscle circumference.
2. Serum albumin and transferrin.
3. Total body nitrogen.
Protein requirement: ICMR recommends 1.0g./kg body weight for Indian adult.
Dietary Goals: AfraTafreeh.com
1. Body weight. Achieve and maintain an appropriate body weight.
2. Total fat. Reduce total calories from fat to no more than 30% of total calories.
3. Saturated fats. Reduce saturated fats to no more than one third of fat intake or less
than 10% of total calories.
4. Monounsaturated fats and polyunsaturated fats. Increase polyunsaturated fats
to no more than 10% of total calories and monounsaturated fats to 10% of calories.
5. Complex carbohydrates. Increase complex carbohydrates to 50 to 60% of total
calories.
6. Fiber. Increase dietary fiber to 20-30 grams per day.
7. Cholesterol. Reduce cholesterol intake to less than 300milli grams per day.
8. Salt. Decrease daily intake to salt to 3 to 8 grams per day.
Nutrition and Disease:

Protein caloric malnutrition are mainly of 3 types.
1. Marasmus.
2. Kwashiorkor
3. Marasmic kwashiorkor.
1. Kwashiorkor: Kwashiorkor is caused by inadequate intake of protein in the
presence of adequate intake of calories.
C/F: oedema, skin lessons, depigmented hair, anorexia, enlarged fatty liver and
decreased plasma albumin.
188 | Biochemistry
2. Marasmus: Results from chronic deficiency of calories.
C/F: arrested growth, extreme muscle wasting, weakness and anaemia.
Diet and Cancer:
a. Dietary fat: High intake of saturated fats are associated with increased risk of cancer
of colon, prostate and breast.
b. Dietary fiber: High fiber diets are associated with lower risk of colon cancer and
diverticulitis.
c. Micronutrients
(i) Cancer of lung has been associated with low intake of vitamin A.
(ii) Stomach cancer is related to deficiency of vitamin C which act by inhibiting
formation of carcinogens nitrosamines in the stomach.
(iii) Selenium has also been implicated in aetiology of cancer.
d. Food additives and contaminants: e.g. preservatives, artificial sweeteners, flavors
has been considered as possible carrinogens.
e. Alcohol: Heavy drinking increases the risk of liver cancer.
Diet and Cardiovascular Disease:
1. Among dietary factors effect of dietary fat, eg. plasma cholesterol and heart disease
is widely accepted.
2. Much stronger correlation exist between LDL-cholesterol and heart disease.
3. High level of HDL-cholesterol has been associated with decresed risk for heart disease.
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Obesity:
1. The basic cause of obesity is overnutrition.
2. A diet containing more energy than needed leads to prolonged post prandial
hyperlipidaeimia and to deposition of triglycerides in adipose tissues resulting in
obesity.
3. Relative insulin resistance takes place in obesity in peripheral tissues, mainly adipose
tissues, while insulin secretion is normal.
Nutrition | 189
Worksheet
 AfraTafreeh.com

190 | Biochemistry
a) Mention RQ of carbohydrate, protein, lipid and mixed diet
b) What are dietary fibers
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Notes:
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Biochemistry Concept Book Atf

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Comprehensive Review Series

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First Published 1999, Delhi Academy of Medical Sciences

© 2021 DAMS Publication

Simple carbohydrates can further be divided into monosaccharide, disaccharide,

High yield points [Direct asked statements]

1) What is the difference in D and L forms

2) What is racemic mixture?

3) What is invert sugar?

High yield points [Direct asked statements]

Hormonal Control on Gluconeogenesis

• Glucagon and epinephrine also stimulate gluconeogenesis but to a somewhat

The main purpose of HMP Shunt pathway is as follow:

Deficiency of various enzyme in glycogen metabolism results in occurrence of various

Glycogenosis Name Cause of disorder Characteristics

Type Ia Von Gierke’s Deficiency of glu-6-phosphatase Hypoglycemia lacticacidemia,

Type Ib –– Endoplasmic reticlum glucose As type Ia; neutropenia and

Type II Pompe’s Deficiency of lysosomal α-1,4 and Fatal, accumulation of glycogen in

Type IIIa Cori’s disease Absence of debranch Accumulation of characteristic

Type IV Andersen’s Absence of branching enzyme Death due to cardiac or liver

Type V McArdle’s Absence of muscle phosphorylase Diminished exercise tolerance;

Type VII Tarui’s disease Deficiency of in msl and RBC As in type V.

Type VIII –– Deficiency of liver Hepatomegaly.

Type IX –– Deficiency of liver and muscle Hepatomegaly.

Type X –– cAMP dependent protein kinese A Hepatomegaly.

Rate limiting step of glycogenolysis Glycogen phosphorylase

Active form of glycogen synthase Dephosphorylated form

Active form of glycogen phosphorylase Phosphorylated form

Label the enzymes at various steps:

Draw the main step and enzymes involved in fructose metabolism

The whole pathway is diagrammatically represented below.

Deficiency of either of the three enzymes results in Galactosaemia, a condition

High yield points [Direct asked statements]

Label the enzymes at various steps:

They are classified as per modified Bloor classification as follow

Phosphatidylcholine Phosphatidic acid

Identify the base in this phospholipid

How acetyl CoA synthesizes fatty acid:

Subsequently fatty acid synthase complex is required, which is multienzyme complex.

Fatty acid chain elongation and denaturation:

High yield points [Direct asked statements]

Acetyl CoA is the starting substrate for synthesis of fatty acid

Stoichiometry of Beta Oxidation:

3. Methylmalonyl CoA is converted to succinyl CoA by methylmalonyl CoA mutase.

Alpha oxidation, is normally a minor pathway and is brought about by hydroxylase

Ketone body and their metabolism

Ketone body synthesis:

Ketone Body Utilization:

High yield points [Direct asked statements]

Metabolic fate of chylomicrons. (A, apolipoprotein A; B-48, apolipoprotein B-48; apolipoprotein C; E,

Reverse Cholesterol Transport and HDL Cycle:

I Lipoprotein lipase deficiency Chylomicrons Triacylgycerols

IIa Hypercholesterolemia LDL Cholesterol

IIb Combined hyperlipidemia LDL, VLDL Triacylglycerols,

III Dysbetalipoproteinemia IDL and CM Remnant Triacylglycerols,

IV Hypertriglyceridemia VLDL Triacylglycerols

V Mixed hyperlipidemia VLDL, chylomicrons Triacylglycerols

High yield points [Direct asked statements]

Mention all dyslipoproteinemias.

Hormonal Effects on Cholesterol Biosynthesis:

1. 7〈-hydroxylation of cholesterol is the first committed step in the biosynthesis

High yield points [Direct asked statements]

Classification of amino acids based on structure