Biochemestry One Liner Unlocked

ONE - LINERS
BIOCHEMISTRY:
for a run through revision
Dr. Smily Pruthi Pahwa

National faculty of PG & FMGE
coaching in biochemistry
MBBS (Sri Guru Ram Das institute of medical sciences
& research, Amritsar, Punjab)
MD Biochemistry (Dayanand medical college
& hospital, Ludhiana, Punjab)
SRship Christian medical college,
Ludhiana, Punjab
Published by: Rakesh Dubey & Rahul Singh
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© Nov 2018
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ACKNOWLEDGEMENT
Although language is the most eloquent vehicle of human expression, yet on the
occasion one finds it difficult to express a feeling appropriately. It is not easy to
formulate in words the feeling of gratitude which I have for many individuals,
who made individual contributions in enabling me to bring this book to
completion.
First and foremost I thank the Almighty for this priceless life and for bestowing
upon his blessings to help me carry out this work with lots of courage and sheer
determination.
Thank you doesn’t seem sufficient for my loving husband Jaspreet Singh whose
invaluable presence is constant source of my happiness and has always endowed
me with strength to face challenges in life. Without his relentless support, this
book would have never been materialized.
Words are insufficient to verbalise my deep sense of gratitude and regards to
my parents, who soulfully provided me the sincere encouragement, endless
patience, emotional and financial support and inspiration throughout my work
and lifting me uphill this phase of life. Special and heartiest thanks to my in laws
for all the support as my parents and encouraging me.
This book would never have been complete without my son Divaahaan who
sacrificed his mother’s time and let me to compose this book.
It is an extraordinary joy to thank my assistant Dimple Nagpal whose
constructive criticism, deep interest and intellectual acumen were instrumental
in accomplishing this book.
I would like to thank M/s Pulse Publications for their efforts and inputs and in
providing me a platform in bringing out the book.
Finally the credit goes to all my students, who have been the actual inspiration
for the composition of this particular book.
vvv
PREFACE
Biochemistry, both a life science as well as a chemical science, is the foundation for
understanding all biological processes. Over the last few decades, a vast information has
been gathered on the subject. It is for sure a standout amongst the most troublesome subjects
as studying and understanding this vast subject is an uphill task for the students. Even after
the students are almost aware about the subject, this subject requires revision.
Keeping all facts in mind, this pocket book aims to enable you to make this tedious work
relatively easy for the students providing the main points to be covered for any entrance
exam containing MCQ type questions on biochemistry. Also the basic theme of the book is
to keep it concise and straightforward.
This book comprises 8 chapters. The first chapter CONCEPTS IN BIOCHEMISTRY
contains the overview of biochemistry and forms the base of student and helps them to
interlink and relate these concepts with other chapters.
The highlights of each topic have been refined and fine tuned to stimulate the learning
process among the students. Several tables and figures are added for the elementary learning.
This book is a time saving hack and comprehends last minute revision notes.
The book is primarily designed for those preparing for various P.G. entrance tests, AIIMS,
AIPGMEE, PGI JIPMER and all India entrance exams and also for undergraduate and
postgraduate medical biochemistry students. This book serves as a ready made source for
MCQ preparation.
In order to score well in these exams, this pocket book is a perfect source for the high time
revision. I have done much justice to providing the best brief knowledge by adding tables,
figures and homophones. I wish you gain maximum from it and develop your concepts.
To help you develop a deeper understanding of the subject, you can have an access to videos
through mobile app “Biochemistry by Dr. Smily Pruthi” in google play store.
Good luck for your coming examinations. Genuine doubts are always welcome. You can be
in touch with me for any doubt at my Facebook group.
Dr. Smily Pruthi Pahwa

M.D. (Biochemistry)
CONTENTS
1. Concepts in Biochemistry ........................................................................ 1-04

2. Carbohydrate Chemistry .......................................................................... 05-10
3. Carbohydrate Metabolism ....................................................................... 11-28
4. Enzymes ..................................................................................................... 29-36
5. Amino Acids ............................................................................................. 37-52
6. Lipids .......................................................................................................... 53-62
7. Molecular ................................................................................................... 63-86
8. Miscellaneous ............................................................................................ 87-102
Colour Images ........................................................................................... 103-108
CHAPTER
CONCEPTS IN
BIOCHEMISTRY 1
CONCEPTS IN BIOCHEMISTRY
z Fed state - When we eat food.
z Fasting state - In between meals, when we are not eating food (Lack of food for12-18
hours).
z Starvation - Severe or complete lack of nutrients (Lack of food since 1-3 days).
z Low Insulin - Glucagon Ratio means Anabolic State.

z High Insulin - Glucagon Ratio means Catabolic State.
z Cyclic AMP causes Phosphorylation. Insulin decreases cyclic AMP and Glucagon
increases cyclic AMP. So Insulin causes Dephosphorylation & Glucagon causes
Phosphorylation.
z Lipoprotein Lipase is activated by Insulin, But only in the capillary beds of Adipose
tissues.
z Hormone Sensitive Lipase is inhibited by Insulin.
z Hormone sensitive lipase is activated by Glucagon and other Insulin Antagonist
Hormones.
z In Fed state, Acetyl CoA is used for the synthesis of Fats or enters TCA.
z In Fasting state, Acetyl CoA has three fates – TCA or Ketone Body Synthesis or
Gluconeogenesis.
z Fats can never be converted to carbohydrates because Link reaction (Pyruvate to
Acetyl CoA) is irreversible. So Acetyl CoA is never Glucogenic i.e. Acetyl CoA can
never be converted to Glucose. But two breakdown products of Fats i.e. Glycerol &
Propionic acid are Glucogenic i.e. they can be converted to Glucose.
z Acetyl CoA activates the very first step of Gluconeogenesis i.e. Pyruvate Carboxylase
(converts Pyruvate to Oxaloacetate).
2 : Concepts in Biochemistry
z Three pathways which occur both in Mitochondria & Cytoplasm are

Gluconeogenesis, Urea cycle & Haem synthesis.
z Pathways which occur in Cytoplasm are : Glycolysis, Glycogenolysis, Glycogenesis,
HMP Pathway, Fatty Acid Synthesis, Cholesterol Synthesis.
z Pathways which occur in Mitochondria are : TCA, ETC, Replication, Transcription
and Translation of mitochondrial DNA, β-Oxidation of Fatty Acids, Ketone Body
Synthesis, Ketone Body Utilization.
z Sources of Blood Glucose are : Food, Liver glycogen (for 12-18 hrs) &
Gluconeogenesis.
z Main fuel for body in Fed state is Carbohydrates.
z Main fuel for body in Fasting state is Fats.
z Heart uses Fatty Acids in fed state.
z Fetal Heart uses Glucose.
z In Heart Failure, fuel for Heart is Glucose.
z Brain cannot use Fatty Acids as Fatty Acids cannot cross Blood Brain Barrier.
z Ketone Bodies are formed during starvation for vital organs – Heart & Brain.
ENZYME CLASSIFICATION
z Enzyme Code or Commission Number 1 is Oxidoreductase.
z Enzyme Code or Commission Number 2 is Transferase.
z Enzyme Code or Commission Number 3 is Hydrolase.
z Enzyme Code or Commission Number 4 is Lyase.
z Enzyme Code or Commission Number 5 is Isomerase.
z Enzyme Code or Commission Number 6 is Ligase.
z If Oxygen is added → known as Oxidation.

z If Hydrogen added → known as Reduction.
z If Electron added → known as Reduction.
z To break a bond, water is added. Hydrolase category uses water to break a bond.
z To make a bond, energy from ATP is used. Ligase category uses ATP to make a bond.
z Lyase category can make a bond, or break a bond, BUT neither uses water nor ATP.
z Transferases are enzymes which transfer a group from one molecule to another.
z Molecular formula is changed when Transferase work.
z Isomerases are enzymes which interconvert various isomers into each other.
z Molecular formula is not changed when Isomerase work.
z Dehydrogenase means removal of Hydrogen means Oxidation.
z If Dehydrogenase enzyme and there is removal of CO2, then it is known as Oxidative
Decarboxylation.
z If Dehydrogenase enzyme and there is removal of NH2 , then it is known as Oxidative
Deamination.
z Reductive Biosynthesis means that in Anabolic Pathway, Reductase enzyme is
required.
Concepts in Biochemistry : 3
z Reductase belongs to EC Number 1 and it requires NADPH.

z NADPH is formed by HMP and two minor sources are Malic enzyme and
Cytoplasmic Isocitrate Dehydrogenase.
z NADPH is used in Reductive Biosynthesis i.e. mainly for Anabolic Pathways e.g.
Fatty Acid synthesis, Cholesterol synthesis, Steroid synthesis, Deoxyribonucleoside
synthesis, required by three aromatic amino acid hydroxylases (Phenylalanine
Hydroxylase, Tyrosine Hydroxylase & Tryptophan Hydroxylase).
z NAD & FAD are used in Catabolic Pathways.
z Coenzyme for Cytoplasmic Isocitrate Dehydrogenase is NADP.
z Coenzyme for Mitochondrial Isocitrate Dehydrogenase is NAD.
z Addition of CO2 is known as Carboxylation. Enzyme is Carboxylase. It requires
ATP, Biotin (Vitamin B7), CO2 & Mg2+ ions.
z Removal of CO2 is known as Decarboxylation. This is of two types : Oxidative
Decarboxylation & Simple Decarboxylation.
z Carboxylation requires vitamin B7 (Biotin).
z Simple Decarboxylation requires vitamin B6 (PLP-Pyridoxal phosphate).
z Oxidative Decarboxylation requires vitamin B1 (Thiamine).
z Thiamine is a Vitamin (vitamin B1).
z Thymine is a Pyrimidine Nitrogenous Base.
z If FAD is used in some reactions, means Vitamin B2 is used.
z If NAD is used in some reactions, means Vitamin B3 is used.
z If CoA is used in some reactions, means Vitamin B5 is used.
z Dehydrogenases are named after the substrate which gets oxidized e.g. Pyruvate
Dehydrogenase.
z Reductases are named after the substrate which gets reduced e.g. Ribonucleotide
Reductase.
z ATP is used when Kinases work. Whenever ATP is used, then Mg is used to stabilize
the outgoing phosphate. Manganese can also be used instead of Magnesium.
z For Substrate Level Phosphorylation, enzyme is always a Kinase.
z For Oxidative Decarboxylation, enzyme is always a Dehydrogenase.
z Few ATPs are derived via Substrate Level Phosphorylation.
z Most ATPs in cells is derived via Oxidative Phosphorylation.
z Cofactor for kinases : Mg++. But Pyruvate Kinase requires K+ more than Mg++.
z Kinase transfer organic phosphate and belongs to Enzyme Code Number: 2.
z Phosphorylase transfer inorganic phosphate and belongs to Enzyme Code Number:2.
z Phosphatase belongs to Enzyme Code Number: 3 (Hydrolase), because this enzyme
removes phosphate by adding water.
z Excess Carbohydrates get converted to Fats in body.
z A person on fat free, carbohydrate rich diet continues to grow obese because of
formation of Endogenous Fat, which is transported in the form of VLDL.
z VLDL transports Endogenous Fat from Liver to Peripheral Tissues.
z Chylomicrons transports Exogenous Fat from Intestine to Peripheral Tissues.
4 : Concepts in Biochemistry
z Atkin’s diet is low calorie, low carbohydrate diet.

z Fructose is the Most Lipogenic Sugar.
z Thermogenic effect of food is also known as Specific Dynamic Action (SDA) is the
amount of energy required to digest, absorb, transport & metabolize the food. This
is maximum for Proteins.
nnn
CHAPTER
CARBOHYDRATE
CHEMISTRY 2
CARBOHYDRATE CHEMISTRY
z Hydroxy group always gives polarity and has high tendency to bind Phosphate.
z Aldehyde group is always present at C-1.
z Keto group is always present at C-2.
z Molecular formula of Carbohydrates is (CH2O)n where n = number of Total Carbons.
z Total number of Isomers possible = 2n , where n = number of asymmetric carbons.
z If all the four valancies of carbon are occupied by different atoms or groups of atoms,
then the carbon is Asymmetric.
z If any two, three or four valancies of carbon are occupied by same atoms or groups
of atoms, then the carbon is Symmetric.
z Functional carbon in case of Carbohydrates (aldehyde or keto) is Symmetric,
But only in Linear configuration. In cyclic configuration functional carbon is
Asymmetric.
z Any compound having asymmetric carbon will show both Optical and Structural
Isomerism.
z Carbohydrates and Amino Acids have asymmetric carbon so both Structural and
Optical Isomerism present.
z Molecules which have Same molecular formula and Different structure are known
as Structural Isomers.
z Molecules which have Same molecular formula and Different optical properties are
known as Optical Isomers.
z Structural Isomerism is of four types → functional isomerism, enantiomerism,
Epimerism and Anomerism.
z Number of asymmetric Carbons in Ketoses are one less the number in aldoses.
z The only carbohydrate with zero asymmetric carbon is → Di Hydroxy Acetone
(DHA).
z Parent alcohol of Carbohydrates → Glycerol.
z Parent carbohydrate → D-Glyceraldehyde.
z Simplest carbohydrate → D-Glyceraldehyde.
z Functional Isomerism - Functional group different (ald or keto).
z Enantiomerism - different -H and -OH orientation around the penultimate carbon.
z Enantiomerism, also known as -D and -L isomerism, also known as mirror images
of each other.
z Enantiomer of carbohydrate abundant in Body/Nature/Plasma/Cell → Always D
form Or For Carbohydrates, it is always ‘D’ form which is abundant.
z Enantiomer of Amino Acid abundant / abundant in Proteins/ synthesized in our
body → L form.
z Enantiomer of a Free Amino Acid in body → Both D and L form exists.
6 : Carbohydrate chemistry
z Examples of D-Amino Acids are: D-serine and D-Aspartate → found in Brain.

z Source of D-amino acid is always exogenous i.e. from diet or from intestinal bacterial
flora.
z Epimerism - different -H and -OH orientation around only one carbon, other than
the penultimate carbon.
z Mannose is an epimer of Glucose at C-2.
z Galactose is an epimer of Glucose at C-4.
z Mannose and Galactose are not epimers of each other, as they differ in two carbons.
z Anomerism - different -H and -OH orientation around the functional carbon, in
cyclic configuration.
z Anomerism exist only in cyclic structures or when carbohydrate is present in
Solution Form.
z In cyclic structures, functional carbon makes a bond with second last carbon.
z Pyranose is a 6 membered ring.
z Furanose is a 5 membered ring.
z Number of carbons in Pyranose Ring = 5 (as one atom is oxygen).
z Number of carbons in Furanose Ring = 4 (as one atom is oxygen).
z Hexoses (6C sugars) can exist as both Pyranose and Furanose.
z Pentoses (5C sugars) can only exist as Furanose.
z Furanose rings are more flexible than pyranose rings, hence selected as components of
nucleic acids. That’s why Nucleic Acids have ribose which always exists as Furanose.
z Two types of Anomers are Alpha (α) and Beta (β).
z α means at functional carbon -OH group is downwards.
z β means at functional carbon -OH group is upwards.
z In most naturally occurring biomolecules, N-glycosidic linkages are in
β-configuration.
z Optical Isomerism → When a plane polarised light is passed through a carbohydrate
solution, then this light gets rotated either towards right (known as dextro-rotatory /
d / ‘+’ sign) or towards left (known as levo-rotatory / l / ‘-’ sign).
z Solution of Glucose is known as Dextrose.
z Glucose is always Dextro-rotatory.
z Fructose is always Levo-rotatory.
z D & L are Enantiomers (Structural Isomerism) but D & L are Optical Isomers
z ‘D’ means OH is towards right side, ‘L’ means OH is towards left side. ‘d’ means
Dextro-rotatory and ‘l’ means Levo-rotatory.
z Racemic mixture is equal d & l (optical isomers). It is optically inactive.
z Racemase enzyme is that enzyme which interconverts D & L into each other
(structural isomers).
z N-Acetyl Neuraminic Acid i.e. NANA (9 carbon sugar/ nanose) is also known as
Sialic Acid.
z Apart from being an important constituent of Gangliosides, Sialic acid is known to
act as Ageing marker for the protein in plasma.
z The proteins devoid of sialic acid are removed from the circulation very fast and
hence have shorter half lives.
z Monosaccharides on oxidation forms Acids.
z Monosaccharides on reduction forms Alcohols.
Carbohydrate chemistry : 7
z Oxidation at C-1 gives Aldonic Acid.

z Oxidation at C-6 or oxidation at primary alcohol gives Uronic Acid.
z Oxidation at both C1 and C6 gives Saccharic Acid.
z Alcohols formed by reduction of Carbohydrates/ monosaccharides are hygroscopic
in nature i.e. they absorb water.
z Type of Cataract in Diabetic patients → Snow Flake Cataract.
z Type of Cataract in Galactosemia patients → Oil Drop Cataract.
z Reducing disaccharides are - Lactose, Maltose, Isomaltose.
z Non-reducing disaccharides are - Sucrose and trehalose.
z Lactose (Milk sugar) - b-1, 4 glycosidic linkage and yields D-galactose and D-Glucose
on hydrolysis.
z All monosaccharides are reducing.
z Molisch Test: general test for Carbohydrates. But one condition → number of
carbons should be 5 or more, only then Molisch’s Test is positive.
z Benedict’s Test: given positive by all reducing sugars.
z Benedict’s Test is a semi-quantitative test i.e. it gives an idea of the quantity of sugar
present.
z Reducing agents such as Uric Acid, Ascorbic Acid, Levodopa, Methyldopa,
Homogentisic Acid, Glutathione and Salicylates can interfere in Urine Analysis
tests- Benedict’s test and Uristix.
z Barfoed’s Test: distinguish between Monosaccharides and Disaccharides. It is given
positive by Reducing Monosaccharides. Positive test is red color. Monosaccharides
react very fast whereas Disaccharides react very slowly. On prolonged heating,
Disaccharides can also give this test positive.
z Seliwanoff’s Test: distinguish between aldehyde and keto sugar. It is given positive
by keto hexose sugars e.g. Fructose.
z Bial’s test: is specific for pentoses (bluish color). Hexoses generally react to give
green, red or brown products.
z Only reducing sugars forms Osazones.
z Osazones of Glucose (Glucosazone crystals), Fructose and Mannose are Needle
Shaped or Broom Stick like appearance.
z Osazones of Galactose (Galactosazone crystals) are like Rhombic Plate.
z Osazones of Maltose (Maltosazone) are Sunflower Shaped.
z Osazones of Lactose have Powder puff or Hedge Hog appearance.
z Routinely used method for blood Glucose estimation-GOD-POD test.
z Iodine test: positive for Polysaccharides which is indicated by formation of blue/
brown/red colour. This color is due to formation of a complex between Iodine & the
Polysaccharide due to adsorption. No change in colour is indicative of presence of
Mono or Disaccharide.
z Starch is the major carbohydrate in diet, which contains Glucose.
z Storage form of carbohydrate in animals in liver and muscle is Glycogen.
z Glycogen is more branched than starch.
z Starch is made up of 20% Amylose (unbranched) and 80% Amylopectin (branched).
z Amylase and Maltase breaks a (1 → 4) bonds.
z Isomaltase breaks α (1 → 6) bonds.
8 : Carbohydrate chemistry
z Malt contains β-Amylase which hydrolyses starch into maltose by sequential

removal of disaccharide units from the non-reducing ends.
z Dextran is a complex, highly branched HMW (high molecular weight)
homopolysaccharide made up of α-Glucose. It is a Storage polysaccharide in yeast
and bacteria.
z Dextran is used as a Plasma Volume Expander in patients of Hypovolemic Shock.
z Commercial name of gel used in Gel Filtration Chromatography is Sephadex.
Biochemically it is dextran.
z Heteropolysaccharides and Cellulose are unbranched.
z Cellulose is the most abundant organic compound in the biosphere and is composed
of repeating disaccharide units of ‘Cellobiose’. Cellobiose is formed by β-glycosidic
linkages between two β-D-Glucose moieties.
z Humans lack enzyme Cellulase which breaks b bonds in cellulose, hence cellulose
acts as fibre in diet.
z Cellulose is not broken due to beta anomerism at C-1.
z Cellulose, Hemi-cellulose and Lignin are Insoluble Fibres and are excreted in its
original form from intestine.
z Pectin and Gums are Soluble Fibres and absorb water thus converting to gel form
and then excreted.
z Soluble fibres are better than insoluble fibres to prevent constipation.
z Honey is rich in fructose.
z Inulin (a homopolysaccharide) is made up of β-fructose.
z Inulin acts as a Prebiotic and is an ideal substance for the measurement of GFR.
z Inulin is not broken due to beta anomerism at C-2.
z Beta bond if broken on one side of galactose then the enzyme is known as
β-Galactosidase. e.g. lactase and Beta Galactosyl Ceramidase (Enzyme deficient in
Krabbe’s disease).
z Heteropolysaccharides are also known as GAGs (Glycosa-Amino-Glycans) as they
contain tandem repeats of amino sugar and uronic acid.
z The only carbohydrate with amino group → Heteropolysaccharide or GAGs
(Glycosa amino glycans).
z GAGs are slimy and slippery because of negative charge given by carboxy, sulphate
and acetate.
z The blood group (A,B,O) substances are Glycosphingolipids.
z Oligosaccharide that is a part of Blood Group ‘O’ substance takes part in the binding
of H. Pylori to the gastric mucosa.
z Hyaluronic acid is the longest GAG.
z No sulphate in hyaluronic acid.
z Heparin is more sulphated than Heparin sulphate and has highest negative charge.
z Heparin acts as an Anticoagulant.
z Heparin Sulphate if accumulated then it is responsible for Mental Retardation.
z Heparin sulphate is a GAG with role in Retinal Cell-Cell Attachment or any cell
cell adhesion.
z Keratin Sulphate does not contain any Uronic acid.
z Keratin sulphate is responsible for Corneal Transparency.
z Cornea has mainly Keratin sulphate and some Chondroitin Sulfate.
Carbohydrate chemistry : 9
z Most widely distributed GAG → Dermatan Sulfate.

z GAG synthesized mainly by Arterial Smooth Muscle Cells - Dermatan Sulfate.
z Dermatan sulphate in excess is responsible for Atherosclerosis.
z Most abundant GAG is Chondroitin Sulfate.
z Major component of Cartilage - Chondroitin Sulfate.
z D-Lyxose is a pentose sugar and is a constituent of ‘Lyxoflavin’ isolated from human
heart muscle.
z The oligosaccharides attached to a number of integral membrane and secretory
glycoproteins are attached to the protein either through the O-linkage to Serine or
Threonine residues or N-linkage to Asparagine residues.
z Chitin - principal component of the hard exoskeletons of Arthropods, consists of
β-1-4 linkage between N-Acetyl-Glucosamine residues.
z Lectins are Carbohydrate Binding Proteins, which play a role in recognition &
attachment.
z Lectins (e.g. Ricin, Hemagglutinin, Cholera toxin) participate in Binding of
Nitrogen Fixing Bacteria, Purification of Glycoproteins and also in Adherence of
E.coli to Intestinal Epithelium.
z Polar Glucose cross the Lipid Bilayer Membrane through Glucose Transporters.
z Phosphorylation of Glucose is done for the Entrapment of Glucose inside the cells.
z SGLT-1 is for Glucose and Galactose Transport in Kidneys and Intestine.
z SGLT-2 is only for Glucose Transport present in Kidneys.
z GLUT-1 is present in Brain, Placenta, Kidneys and also in RBCs.
z GLUT-3 is present in Brain, Placenta, Kidneys.
z GLUT-1 and GLUT-3 are active during Fasting state whereas GLUT-2 is active
during Fed state.
z Hypoglycemic action of Insulin is via GLUT-4.
z Only GLUT-4 is Insulin Dependent as it recruits GLUT-4 on the membrane of
peripheral cells.
z GLUT-1 is most abundant in RBCs.
z Major GLUT in Neurons is GLUT-3.
z Rate at which Glucose (solute) enters a cell via GLUT (Facilitative Transport)
depends upon :
1) Number of GLUTs
2) Concentration of Glucose
3) Affinity of GLUT for Glucose
z Glucose is the main energy source for Brain, RBCs, Retina, Cornea, Renal Medulla,
Testis.
z RBCs can use only Glucose (in Fed/Fasting/Starvation).
z Glucose is the Universal Fuel for Fetus.
nnn
CHAPTER
CARBOHYDRATE
METABOLISM 3
GLYCOLYSIS
Glucose
Hexokinase ATP
1 ADP
Glucose 6-phosphate
2 Phosphoglucose
isomerase
Fructose 6-phosphate
3 Phosphofructo- ATP
kinase-1 ADP
Fructose 1,6-bisphosphate
4 Aldolase-A
Triose
5 phosphate Glyceraldehyde
Dihydroxyacetone isomerase
phosphate 3-phosphate
(2 molecules)
Glyceraldehyde +
2 NAD + 2P1
6 3-phosphate 2 NADH + 2H
+
dehydrogenase
1,3-Bisphosphoglycerate
(2 molecules)
Phosphoglycerate 2 ADP
7 kinase 2 ATP
3-phosphoglycerate
(2 molecules)
Phosphoglycero-
8 mutase
2-phosphoglycerate
(2 molecules)
9 Enolase
Phosphoenolpyruvate
(2 molecules)
10 Pyruvate 2 ADP
kinase 2 ATP
NADH NAD
Pyruvate Lactate
(2 molecules) (2 molecules)
11 Lactate Dehydrogenase
(anaerobic)
12 : Carbohydrate Metabolism
z Also known as Embden Meyerhoff Pathway/EMP pathway.

z Glycolysis is activated by Insulin.
z Intracellular site for operation of Glycolysis : Cytosol.
z Occurs in all cells of the body.
z Occurs in both Aerobic and Anaerobic conditions.
z Glucose is the only molecule which can produce ATP without O2.
z Glycolysis is not the complete breakdown of Glucose.
z Glycolysis is irreversible.
z Steps 1 to 5 in Glycolysis constitute Phase I, also known as Energy Utilizing Phase
as 2 ATPs are used.
z Steps 5 to 10 constitute Phase II of Glycolysis, also known as Energy Producing
Phase.
z Glucokinase/Hexokinase IV is induced by Insulin.
z Glucokinase has specificity for D-Glucose.
z Hexokinase has Feedback Inhibition from the product Glucose-6-Phosphate.
z Hexokinase is Flux Generating Step of Glycolysis.
z Phosphorylation of Fructose-6-Phosphate to Fructose 1,6 Bisphosphate is
Committed Step of Glycolysis i.e. enzyme PFK-1 (Phospho Fructo Kinase-1) the
Rate Limiting Enzyme.
z ATP is Negative Homotropic Regulator of PFK-1.
z Citrate is Negative Heterotropic Allosteric Regulator of PFK-1.
z Inhibition of Glycolysis by Citrate directs Glucose for Glycogen Synthesis
z AMP & Fructose 2, 6 Bisphosphate are Positive Homotropic Allosteric Effectors
of PFK-1.
z Presence of ATP (indicates high energy state) inhibits PFK-1 and presence of ADP
(indicates low energy state) activates PFK-1.
z Aldolase A belongs to Lyase category as it breaks Fructose 1, 6 Bisphosphate into two
3C molecules i.e. Glyceraldehyde-3-P and Dihydroxy Acetone Phosphate (DHAP)
without using H2O.
z Aldolase B – a hepatic isoform is involved in Fructose Metabolism.
z Glyceraldehyde-3-Phosphate Dehydrogenase is the only enzyme of Glycolysis
which can use Inorganic Phosphate.
z Glyceraldehyde-3-P Dehydrogenase is inhibited by Iodoacetate and Arsenate.
z Conversion of Glyceraldehydes-3-P to 1, 3 Bisphospho Glycerate is the first
Oxidation-Reduction step of Glycolysis.
z In Anaerobic Glycolysis, ATP is formed by Substrate Level Phosphorylation and
not Oxidative Phosphorylation.
z Irreversible or Regulatory Steps of Glycolysis are → Glucokinase, PFK-1 and
Pyruvate Kinase.
z Substrate Level Phosphorylation (SLP) steps of Glycolysis are Phospho Glycerate
Kinase and Pyruvate Kinase.
z All SLP steps are reversible except Pyruvate Kinase which is irreversible.
z In Glycolysis, 7 steps are reversible, which are also used by Gluconeogenesis.
z Mutase used in Glycolysis is Phospho-Glycero Mutase but Mutase used in Glycogen
Metabolism is Phosho-Gluco Mutase.
z Enolase is inhibited by Sodium Fluoride, which is used in Blood Glucose estimation.
Carbohydrate Metabolism : 13
z Pyruvate is the end product in Aerobic Glycolysis.

z Excessive formation of Lactic Acid can be prevented by inhibiting PFK-1.
z Insulin activates Glucokinase, PFK-1 and Pyruvate Kinase.
z Pyruvate Kinase deficiency is the second most common Human Enzyme Deficiency.
In this Haemolysis occurs because RBCs rely only on Glucose and due to lack of
energy in RBCs, Sodium-Potassium Pump of RBCs is affected.
z Increased PFK-1 increases Fructose1,6 Bisphosphate which activates Pyruvate
Kinase.
z Pyruvate Kinase is activated by AMP & Fructose 1,6 Bisphosphate.
z The extra step of Anaerobic Glycolysis occurs for the Replenishment of NAD.
z Lactate is the Dead End of Glycolysis.
z Lactate Dehydrogenase (LDH) is inhibited by Oxamate.
z Fates of Glucose-6-P are HMP, Glycogenesis and Gluconeogenesis
z Number of ATP produced in RBCs in Fed state, Fasting state, Aerobic state and
Anaerobic state is 2 ATP.
z Rapaport Leubering (RL) Shunt occurs only in RBCs.
z Phosphoglycerate Kinase step is bypassed in RL Shunt.
z Number of ATPs in RBCs are never more than 2 , but it can be less than 2 if it is RL
(Rapaport Leubering) Shunt.
z PFK-I forms Fructose 1, 6 Bisphosphate.
z PFK-II forms Fructose 2, 6 Bisphosphate.
z Product of PFK-II (Fructose 2,6 Bisphosphate) activates PFK-I.
z Fructose 2, 6 Bisphosphate is formed in Fed state & it activates Glycolysis but it
decreases Gluconeogenesis.
LINK REACTION
Cytosol Mitochondrion
TRANSPORT PROTEIN
(Pyruvate-Proton NAD+ NADH + H+
CO2
Symport)
PYRUVATE Pyruvate Acetyl CoA

Pyruvate (2C)
(3C) Dehydrogenase
Complex
Coenzyme A
IRREVERSIBLE
Oxidative 5 coenzymes required
Decarboxylation are : Lipoic acid, Vitamin
B1, B2, B3, B5
z Link reaction (Conversion of Pyruvate to Acetyl CoA) is a link between Glycolysis

& TCA.
z Occurs in Mitochondrial Matrix
z Link reaction step is Oxidative Decarboxylation (Requires Vitamin B1)
z Occurs in Fed state
z Link reaction is activated by Insulin
z Pyruvate enters Mitochondria via Pyruvate Proton Symport present in the Inner
Mitochondrial Membrane
z Link reaction enzyme is Pyruvate Dehydrogenase complex (PDH complex)
z PDH complex is a Multienzyme containing three components:
1) E1-Pyruvate Dehydrogenase or Pyruvate Carboxylase
2) E2-Dihydro Lipoyl Transacetylase
3) E3-Dihydro Lipoyl Dehydrogenase
z PDH complex and TCA both requires five coenzymes – Vitamin B1, B2, B3, B5 and
Lipoic acid
z Deficiency of these Vitamins (B1, B2, B3, B5) leads to decreased energy production
and CNS problems as Brain cells depend upon this Aerobic reaction
z Allosteric Inhibition of PDH occurs by Acetyl CoA, NADH and ATP
z PDH is active in Dephosphorylated state and Dephosphorylation is done by Insulin
z Arsenate inhibits the enzymes requiring Lipoic Acid as coenzyme such as PDH
complex
z Deficiency of PDH complex is the most common biochemical cause of Congenital
Lactic Acidosis.
z Beri-Beri causes Lactic acidosis
z Link reaction is irreversible.
TCA CYCLE
Acetyl CoA
Malate Oxaloacetate
Citrate synthase
dehydrogenase
NADH
NAD
Malate Citrate
Fumarase Aconitase
Fumarate Isocitrate
FADH NAD
Succinate Isocitrate
dehydrogenase FAD dehydrogenase
NADH
CO2
Succinate -ketoglutarate
GTP NAD
GDP
NADH
Succinyl -ketoglutarate
thiokinase Succinyl-CoA dehydrogenase
CO2
z TCA is both Anabolic & Catabolic i.e. it is Amphibolic.

z TCA is a Vital cycle, it has no Hormonal Control.
z TCA cannot occur in Anaerobic conditions and is strictly Aerobic.
z Occurs both in Fed as well as Fasting state.
z Neither activated by Insulin nor Glucagon.
z Occurs in all cells of body where Mitochondria is present (occurs in Mitochondrial
Matrix).
z TCA known as Tricarboxylic Acid Cycle as Citrate and Isocitrate are Tricarboxylic
Acids.
z Kreb’s cycle name is by name of scientist ‘Hans Adolf Krebs’.
z TCA is also called Citric Acid cycle as first compound formed is Citrate which is a
Prochiral molecule.
z Prochiral Molecules are those which can be converted from Achiral to Chiral in a
single step.
z Citrate inhibits Glycolysis and activates Fatty Acid Synthesis.
z Four steps involved in Oxidation-Reduction (Enzymes involved are Isocitrate
Dehydrogenase, α-KG Dehydrogenase, Succinate Dehydrogenase and Malate
Dehydrogenase.
z Two steps involved in Oxidative Decarboxylation (Isocitrate DHase and α-KG
DHase).
z Oxaloacetate (OAA) is regarded as First Substrate and Carrier of TCA and not
Acetyl CoA.
z Oxaloacetate has a Catalytic role in TCA.
z Acetyl CoA is not the intermediate of TCA and is never Glucogenic.
z Aconitase always acts on the part of Citrate derived from Oxaloacetate.
z Flouroacetate is Non Competitive Inhibitor of Aconitase.
z Flourocitrate is Competitive Inhibitor of Aconitase.
z Aconitase/Aconitate Hydratase belongs to Lyase category and is exceptionally an
Iron-Sulphur Protein.
z α-KG Dehydrogenase is a Multi Enzyme Complex similar to PDH complex and
requires same five coenzymes (TPP, Lipoic Acid, FAD, CoA, NAD)
z α-KG Dehydrogenase is not regulated by Phosphorylation or Dephosphorylation.
z Enzymes catalyzing Dehydrogenation without Decarboxylation do not require
Metallic ions for their activity (Mg2+, Mn2+).
z Substrate level Phosphorylation Step : Conversion of Succinyl CoA to Succinate
by Succinyl CoA Synthetase/ Succinate Thiokinase (produce both ATP & GTP but
ATP is predominant) GTP is produced in Liver and Kidney during Starvation for
PEPCK enzyme of Gluconeogenesis.
z There is only one Substrate Level Phosphorylation step in TCA.
z All enzymes of TCA lies in Mitochondrial Matrix Except Succinate Dehydrogenase
(located in Inner Mitochondrial Membrane).
z Succinate Dehydrogenase is a Flavoprotein Enzyme in which FAD act as Hydrogen
Acceptor and is inhibited by Malonate.
z Succinate Dehydrogenase is involved in TCA & ETC.
z Fumarate Hydratase/Fumarase and Aconitase are Lyases.
z One Acetyl CoA gives 10 ATPs in TCA cycle (9 by Oxidative Phosphorylation/ETC
and 1 by SLP).
z Anaplerotic Reactions are those reactions which replenish TCA intermediates.
z Most important Anaplerotic reaction is Pyruvate to Oxaloacetate conversion by
Pyruvate Carboxylase.
z Two CO2 in TCA are derived from the Carbons of Oxaloacetate.
z Irreversible steps of TCA are Citrate Synthase & α-Ketoglutarate Dehydrogenase.
z There are three Rate Limiting Enzymes in TCA depending on various complex
conditions in body : Citrate Synthase, Isocitrate Synthase & α-Ketoglutarate
Dehydrogenase
WARBURG’s EFFECT
z It occurs in Cancerous Cells. CANCER
z Aerobic Glycolysis occurs in Glycolysis CELL
Cancerous cells but End Product is
Lactate.
z One Glucose in Cancerous cells Pyruvate Lactate
gives only 2 ATPs. Oxygen
z Warburg’s effect is Aerobic
Glycolysis and no Oxidative
Phosphorylation.
SHUTTLES
z Transport of NADH from Cytoplasm to Mitochondria occurs with the help of two
Shuttles namely Malate Shuttle and Glycerol Phosphate Shuttle.
z NADH cannot cross Inner Mitochondrial Membrane (IMM). So Shuttles are used to
transport NADH from Cytoplasm to Mitochondria.
z NADH Shuttles (Malate Shuttle and Glycerol Phosphate Shuttle) are only used by
those Pathways which occur in Cytoplasm.
z Malate Shuttle is important

in Liver and Heart.
z Cytoplasmic Malate
Dehydrogenase (MDH)
converts Oxaloacetate to
Malate and uses NADH.
z Malate, Pyruvate &
Aspartate can cross Inner
Mitochondrial Membrane.
Malate-Aspartate Shuttle
z Mitochondrial Malate Dehydrogenase (MDH) converts Malate to Oxaloacetate

and produces NADH.
z Oxaloacetate cannot cross IMM.
z Malate Shuttle is also known as Malate-Aspartate Shuttle as Malate and aspartate
cross IMM in this shuttle.
z Glycerol-P-shuttle is more important in Brain and Skeletal Muscles.
z Cytoplasmic Glycerol-3-P Dehydrogenase enzyme converts Dihydroxy Acetone
Phosphate (DHAP) to Glycerol-3-Phosphate and uses NADH.
z Mitochondrial Glycerol-3-P dehydrogenase converts Glycerol-3-P to Dihydroxy
Acetone Phosphate (DHAP) and uses FAD+.
NAD+ Glycerol-3P FAD
Cytoplasmic Mitochondrial
glycerol-3-P DH glycerol-3-P DH
+
NADH+H DHAP FADH2
Cytosol Inner mitochondrial Mitochondrial

membrane matrix
Glycerol-3-Phosphate Shuttle
z Malate Shuttle takes NADH from Cytoplasm and delivers NADH in the
Mitochondria.
z Glycerol Phosphate Shuttle takes NADH from Cytoplasm and delivers FADH2 in
the Mitochondria.
z Glycerol Phosphate Shuttle is a shorter shuttle and is a quick source of ATPs and is
less prone to be affected by any deficiency.
z Citrate Shuttle is used in Fatty Acid Synthesis.
z Citrate Shuttle transfers Acetyl CoA from Mitochondria to Cytoplasm.
z ATP Citrate Lyase breaks Citrate to Oxaloacetate and Acetyl CoA in Cytoplasm.
z Creatine-Phosphate Shuttle transport ATP from Mitochondria to Cytoplasm.
z ADP-ATP Translocase present in IMM transfers ATP out into intermembrane
space and ADP transferred inside into the Mitochondrial Matrix.
z Enzyme Creatine Kinase –Mitochondrial (CKm) in Intermembrane Space converts
Creatine to Creatine Phosphate and this Creatine Phosphate reaches Cytoplasm.
z Cytoplasmic Creatine Kinase again converts Creatine Phosphate to Creatine and
this phosphate is used to form ATP.
ATP ATP ATP

ETC/ ELECTRON TRANSPORT CHAIN
z NADH is the main starting material for ETC, also sometimes FADH2.
z NADH enters ETC and gives 2.5ATPs.
z FADH2 enters ETC and gives 1.5ATPs.
z ETC & TCA are Vital Pathways for cells and both occur in Mitochondria.
z ETC occurs in IMM, therefore, all components lie in IMM.
z ETC occurs in all cells of the body where Mitochondria is present.
z ETC occurs only in Aerobic conditions.
z ETC occurs in Fed and Fasting state.
z ETC has 5 Protein Complexes- Complex I, II, III, IV, V.
z Complex I to IV are involved in Electron Transport.
z Complex I is also known as NADH-CoQ Reductase / NADH Dehydrogenase.
z Complex II is also known as Succinate CoQ Reductase.
z Complex III is also known as Cyt C Reductase or CoQ-Cytochrome c Reductase
or Cytochrome b/c1.
z Complex IV is also known as Cyt C Oxidase or Cytochrome a/a3 (Prosthetic
Group-Cu).
z Complex IV is the only Electron Carrier in which Haem Iron has an available
coordination site that can directly react with O2.
z Complex V has two units:
1) F0- present in IMM and is a Proton Channel.
2) F1- protrudes into Mitochondrial Matrix and is having enzyme activity of ATP
Synthase.
z ATP Synthase is also known as F1/F0-ATPase because it can also catalyse the
hydrolysis of ATP to ADP and Pi.
z Coenzyme Q (also known as Ubiquinone) is the only Non-Protein member of ETC.
It is a Lipid Soluble Quinine and also an Anti-Oxidant.
z Cytochrome c is the only member of ETC, not lying in IMM.

z Redox Potential is the tendency to gain electrons.
z NADH has minimum Redox Potential (Strong Electron Donor).
z O2 has maximum Redox Potential (Strong Electron Acceptor).
z Electrons Flow occurs from NADH to O2 in Electron Transport Chain.
z Total 10 H+ are responsible for providing 2.5 ATPs from 1 molecule of NADH.
z Complex I and Complex III transfers 4H+, so they are responsible for the production
of 1 ATP each.
z Complex IV transfers 2H+, so it is responsible for production of 0.5 ATP.
z When ETC starts from FADH2, FADH2 gives electrons to Succinate Dehydrogenase,
which further gives to Complex II and then to CoQ.
z Complex II does not form any ATP.
z In ETC, Oxidation and Phosphorylation are coupled i.e. they always occur together.
z Uncoupling means Oxidation is occurring but Phosphorylation does not occur.
z A substance which creates a hole in the IMM acts as an Uncoupler.
z Uncouplers – DNP (2,4 Dintrophenol), Thermogenin (known as Uncoupling
protein-1/UCP-1), Thyroxine and Free fatty acids.
z Thermogenin is a protein present in Brown Fat and is responsible for Non-Shivering
Thermogenesis. So Brown Fat is involved in Energy Expenditure
z ADP to ATP Conversion or Complex V is inhibited by Oligomycin. It inhibits both
Oxidation and Phosphorylation.
z ADP to ATP transfer is inhibited by Atractyloside. Enzyme inhibited is ADP-ATP
Translocase.
z Malonate (3C) is inhibitor of Complex II of ETC, Succinate Dehydrogenase of TCA
cycle and CPT-I (Carnitine Palmitoyl Transferase-I) of β-Oxidation of Fatty Acids.
z Rotenone and Phenobarbitone inhibits Complex I of ETC.
z Phenformin inhibits Complex III of ETC.
z Cyanide (CN), Carbon Monoxide (CO), Hydrogen Sulphide (H2S) inhibits
Complex IV of ETC.
z Increased NADH leads to Lactic Acidosis and Hyperuricemia.
GLUCONEOGENESIS
z Occurs in both (2) Pyruvate
2 ATP
Mitochondria and Pyruvate carboxylase
Cytoplasm (starts from 2 ADP
Mitochondria) (2) Oxaloacetate

2 GTP
z Organs: 90% in Liver PEP Carboxy Kinase
(predominant) and 10% in 2 GDP
Kidneys. (2) PEP
z Gluconeogenesis occur
during Fasting/Starvation
(2) 3-Phosphoglycerate
and in Diabetes Mellitus 2 ATP
z First step of Gluconeogenesis Phospho Glycerate Kinase
2 ADP
is conversion of Pyruvate to
(2) 1,3-Bisphosphoglycerate
Oxaloacetate by Pyruvate NADH + H+
Carboxylase enzyme +
NAD
(Anaplerotic reaction).
Fructose-1,6-bisphosphate
z Acetyl CoA is very important
Allosteric Activator of P1 Fructose1,6 bisphosphatase
Pyruvate Carboxylase.
Fructose-6-phosphate
z Oxaloacetate produced in
Mitochondria gets converted
to Malate by Mitochondrial Glucose-6-phosphate
Malate Dehydrogenase as
P1
Oxaloacetate cannot cross Glucose-6-Phosphatase
IMM. Glucose
z Malate cross IMM and is again converted to Oxaloacetate by Cytoplasmic Malate

Dehydrogenase.
z Phospho Enol Pyruvate Carboxy Kinase (PEPCK) converts Oxaloacetate to
Phosphoenol Pyruvate which gets converted to Fructose1,6 Bisphosphate.
z Enzymes common in Glycolysis and Gluconeogenesis: Enolase, Phosphoglycero
Mutase, Phospho Glycerate Kinase, Glyceraldehyde-3-P-Dehydrogenase and
Aldolase A.
z All the bypass reactions of Gluconeogenesis are irreversible.
z Gluconeogenesis is not just the reversal of Glycolysis as three enzymes are different
in Gluconeogenesis.
z Instead of Pyruvate Kinase in Glycolysis, Enzymes used in Gluconeogenesis are
Pyruvate Carboxylase and PEPCK.
z Instead of PFK-1, Fructose 1,6 Bisphosphatase is used in Gluconeogenesis
z Instead of Hexokinase, Glucose-6-Phosphatase is used in Gluconeogenesis
z Glucose-6-Phosphate of Hepatocyte is not acted upon by Glucose-6-Phosphatase
as soon as it is formed because Glucose -6-Phosphatase enzyme is present in the
Membrane of ER, not in the Cytoplasm.
z Sequence of Compartments for Gluconeogenesis : Mitochondria → Cytoplasm → ER.
z Glucose-6-Phosphatase is common enzyme in Gluconeogenesis and Glycogenolysis.

z Rate Limiting Enzymes for Gluconeogenesis are Pyruvate carboxylase, PEPCK and
Fructose 1,6 Bisphosphatase depending upon various complex situations in the body.
z Substrates of Gluconeogenesis are Pyruvate, Lactate, Glycerol, Propionic Acid,
Any TCA intermediate and Amino Acids (Glucogenic category & both Glucogenic
–Ketogenic category).
z Purely Ketogenic Amino Acids are Leucine and Lysine (if both given mark Leucine
as Lysine is in controversy).
z Both Glucogenic and Ketogenic Amino Acids are Tyrosine, Tryptophan, Threonine,
Isoleucine and Phenylalanine (Total Are Five).
z Total Ketogenic Amino Acids = 7 (2 are purely Ketogenic and 5 are in both category).
z Total Glucogenic Amino Acids = 18 (13 are purely Glucogenic and 5 are in both
category).
z So only 2 Amino Acids can never form Glucose. Rest 18 can form Glucose
z Mostly 3-C containing compounds are Good Substrates for Gluconeogenesis e.g.
Pyruvate, Lactate, Alanine.
z Link Reaction (Pyruvate Dehydrogenase complex) is inhibited during Fasting,
so excess of Pyruvate which is not entering into Link Reaction is used up for
Gluconeogenesis.
z If Fructose 2,6 Bisphosphatase is active, Glycolysis decreases.
z Fructose 2,6 Bisphosphatase activity is present in TIGAR (TP-53 Induced Glycolysis
& Apoptosis Regulator).
z Two Pyruvate to Glucose formation by Gluconeogenesis uses 6 ATPs (or 4 ATPs
& 2 GTPs).
z Two Lactate to Glucose formation by Gluconeogenesis uses 6 ATPs (or 4 ATPs &
2 GTPs).
z Two Alanine to Glucose formation by Gluconeogenesis uses 6 ATPs (or 4 ATPs &
2 GTPs).
z In Gluconeogenesis, ATPs are used by Pyruvate Carboxylase and Phospho Glycerol
Kinase steps and GTP is used by PEPCK enzyme.
GLYCOGEN
z Glycogen is the Storage form of The liver
Carbohydrates. stores
glucose as
z Glucose is stored as Glycogen glycogen
because it packs a large quantity
of Carbohydrates in small space,
Glycogenesis Glycogenolysis
maintains low osmolarity in cells
and interconversion of Glucose
and Glycogen provides a good
Regulatory Pathway. Blood
Glucose
z During exercise, Anaerobic conditions prevail and as Fats cannot be metabolized

Anaerobically, only Glucose can be used which is obtained from Glycogen.
z Mobilization of Glycogen is faster than Fats.
z Fats can never be converted to Glucose so Fats cannot maintain Blood Glucose levels.
z Fatty acids cannot cross Blood Brain Barrier but Glucose can.
z Glycogenin is a Glycoprotein which acts as a Primer in Glycogen synthesis
z Glycogenin also act as Auto-Catalyst (in attaching Glucose on Glycogenin)
z Glucose gets attached to –OH group of Tyrosine in Glycogenin.
z Synthesis of Glycogen from Glucose is known as Glycogenesis.
z Glycogenesis is Anabolic Pathway and hence activated by Insulin and occurs in Fed
state.
z Glycogen Synthase activity is depressed by cyclic AMP.
z In Glycogen, Straight chains have α (1→ 4) bonds and the Branching point has α
(1 → 6) bond.
z Two Glucose residues are joined by α (1→4) bond.
z Formation of UDP-Glucose is carried out by UDP-Glucose Pyrophosphorylase.
z Glycogen Synthase adds Glucose residues to the non-reducing end of Primer.
z Only straight chains are synthesized by Glycogen Synthase.
Minor glycogenolysis 2. Pompe’s

4. Anderson Acid Maltase
Glycogen
Branching enzyme 5. Mc Ardle’s (Ms)
6. Her’s (Liver)
1->4 glucose units 1. Glycogen
Glycogen Synthase Phosphorylase (RLE)
RLE 2. Glucan Transferase
UDP glucose 3. Debranching enzyme
UDP glucose
pyrophosphorylase
3. Cori’s/Fobe’s/
UTP
Glucose-1-P Limit Dextrinosis
PPi Mutase
1. Von Gierke’s
Glucose-6-P
Hexokinase/
Glucose-6-Pase
Glycogenesis Glucokinase Glycogenolysis
Glucose
z Branching enzyme is also known as Amylo α (1 → 4) (1 → 6 ) transglycosylase or

4:6 transferase.
z The branched structure of Glycogen permits the rapid release of Glucose
simultaneously from every Non Reducing End of every branch.
z Breakdown of Glycogen to produce Glucose is called Glycogenolysis.
z Glycogenolysis is Catabolic Pathway and hence activated by Glucagon and occurs in
Fasting state or in between meals.
z By weight, Glycogen is more in Liver (means when we take same weight of Liver &
Muscle, then it is more in Liver).
z By percentage, Glycogen is more in Muscle (means when we calculate total body
percentage Glycogen, then it is more in Muscle as Muscle Mass is more).
z In Liver, the function of Glycogen is to

maintain Normal Glucose levels.
z End product of Liver Glycogenolysis is
free Glucose as Glucose-6-Phosphatase
enzyme is present.
z Liver Glycogen stores are increased in Fed
state and depleted in Fasting.
z In Muscle, the function of Glycogen
is to give energy for Muscle activity/
contraction.
z Skeletal Muscle does not contribute Glucose to blood as Glucose-6-Phosphatase

enzyme is absent. So, End product of Muscle Glycogenolysis is Glucose -6-Phosphate.
This Glucose-6-P directly enters Glycolysis.
z Muscle Glycogen stores not affected in short period and are moderately decreased
in prolonged Fasting.
z Minor pathway of Glycogenolysis occurs in Lysosomes (1-3%). Enzyme is Acid
Maltase or Acid α (1 → 4) Glucosidase. Deficiency of this enzyme is known as Type
II Glycogen storage disease (Pompe’s disease).
z Pompe’s disease or Type II GSD is the only Glycogen Storage disease, which is a
Lysosomal Storage Disease.
z Enzymes involved in major pathway of Glycogenolysis are Glycogen Phosphorylase,
Glucan Transferase and Debranching enzyme.
z Glycogen Phosphorylase (RLE) breaks α (1 → 4) bonds, starting from non reducing
end, till four Glucose residues are left at the branch point.
z Glucose is liberated in the form of UDP-Glucose from Glycogen.
z Glycogen Phosphorylase (RLE of Glycogenolysis) is active in phosphorylated state.
z Activators of Glycogen Phosphorylase are cyclic AMP, Glucagon, Epinephrine/
Nor-Epinephrine, Calcium- Calmodulin, Kinase and 5’AMP.
z Epinephrine acts in Muscle and Liver but Glucagon acts only in Liver.
z Inhibitors of Glycogen Phosphorylase are Protein Phosphatase, Insulin, Glucose,
Glu-6-Phosphate, ATP and Fruct-6-Phosphate.
z Glucan Transferase or oligo α (1 → 4): α(1 → 4) Glucan Transferase or 4:4
Transferase transfers 3 Glucose residues from one chain to the neighbouring chain,
but does not releases any Glucose
z Debranching Enzyme (amylo α (1 → 6) Glucosidase activity) is a bifunctional
enzyme having two enzymatic activities on a single protein i.e. Glucan transferase
and Debranching enzyme.
z Primary end product of Glycogenolysis obtained by breaking α (1 → 4) bonds is
Glucose-1-Phosphate.
z Synthesis of Glycogen from Non Carbohydrate Sources is known as Glyconeogenesis
(Same as Gluconeogenesis except the last step of breakdown of Glucose-6-P not
occurring and Glucose-6-P directly goes for Glycogen formation).
z Both Glycogenesis and Glycogenolysis occurs in Cytoplasm.

z Glycogen Synthesis and Breakdown occurs in Liver as well as in Muscle.
z Both Rate Limiting Enzymes of Glycogen Metabolism are Transferases.
z Free Glucose can cross Cell Membrane via GLUTs but Phosphorylated Glucose
(Glucose-6-P is entrapped inside the cells.
z Enzyme common between Glycolysis and Glycogenesis – Hexokinase/Glucokinase.
z Enzyme common between Glycogenesis and Glycogenolysis – Phospho-Gluco-
Mutase.
z Enzyme common between Glycogenolysis and Gluconeogenesis – Glucose-6-
Phosphatase.
z Most Glycogen storage disease (GSD) affect Glycogenolysis.
z Most common GSD – Type I GSD known as Von Gierke’s Disease and the enzyme
deficient is Glucose-6-Phosphatase.
z Type I GSD clinical features are Severe Hypoglycaemia, Ketosis, Enlarged Liver and
Kidneys, Hypertriglyceridemia, Lactic Acidosis and Hyperuricemia
z Causes of Hyperuricemia in Von Gierke’s Disease are Lactic Acidosis, HMP and
Sequestration of Pi.
z Type III GSD known as Cori’s Disease/Limit Dextrinosis and the enzyme deficient
is Debranching enzyme.
z Type IV GSD known as Anderson’s Disease/Amylopectinosis and the enzyme
deficient is Branching enzyme.
z Type V GSD known as Mc Ardle’s Disease and the enzyme deficient is Muscle
Phosphorylase.
z Type VI GSD known as Her’s Disease and the enzyme deficient is Liver
Phosphorylase.
MINOR PATHWAYS OF CARBOHYDRATES :- HMP, Uronic Acid Pathway, Fructose

Metabolism, Galactose Metabolism
HMP (Hexose Mono Phosphate Pathway)
z HMP- Hexose Mono Phosphate

Pathway because Glucose-6-P is Glycolysis
the starting material Glucose
z HMP is also known as PPP
(Pentose Phosphate Pathway) Glucose-6-Phosphate
because Ribose-5-P is synthesized Pentose
Phosphate
by this pathway. Pathway
z HMP is activated by Insulin. Citric Acid Cycle
z Intracellular site for HMP is
Cytosol.
z The only source of Ribose-5-P is Electron Transport Chain
HMP.
z Occurs in Liver, Adipose Tissue, Lactating Mammary Glands, Adrenal Cortex,

Gonads and RBCs.
z HMP is the minor pathway for oxidation of Glucose but a major source for NADPH.
z NADPH produced in HMP is needed for Reductive Biosynthesis or to counter the
damaging effects of Oxygen Radicals.
z No ATP is generated but CO2 is produced in HMP.
z HMP has 2 phases: Oxidative phase (irreversible) and Non-Oxidative phase
(reversible).
z In cells with greater need for NADPH than Ribose-5-P, both Oxidative and Non-
Oxidative Phase occurs.
z In cells with greater need for Ribose-5-P than NADPH, only Non-Oxidative Phase
occurs.
z Oxidative Phase synthesize NADPH.
z Non-Oxidative Phase synthesize Ribose-5-Phosphate.
z Heptose keto sugar formed in HMP shunt is Sedoheptulose-7-P.
z Glucose-6-P dehydrogenase (G-6-PD) catalyzes the conversion of Glucose-6-P to
6-Phosphogluconate.This is the Rate Limiting step, first Commited step and also the
Regulatory step of HMP.
z NADPH is a potent Competitive Inhibitor of Glucose-6-P dehydrogenase.
z Insulin upregulates the expression of gene for Glucose-6-P dehydrogenase
z Pathways which do not produce ATP are HMP, Uronic Acid Pathway and RL shunt,
Synthesis of Ketone Bodies, α-Oxidation of Fatty Acids & Oxidation of VLCFA
(Very Long Chain Fatty Acids).
z NADPH is produced from : HMP (major source), Malic enzyme and Cytosolic
Isocitrate Dehydrogenase.
z In Oxidative Phase, 2 molecules of NADPH, 1 molecule of CO2 and 1 molecule of
Ribulose-5-P is formed per Glucose-6-P molecule oxidized.
z Products of HMP from three molecules of Glucose-6-P are 6 NADPH, 3 CO2 , 2
molecules of Glucose-6-P and one Glyceraldehyde-3-P.
z Transketolase → requires TPP (derivative of Vitamin B1) and Mg++.
z Transketolase transfers 2 carbon units.
z Transaldolase transfers 3 carbon units.
z Molecule which is Intermediate as well as End Product in HMP - Glyceraldehyde-
3-Phosphate.
z Molecule which is Substrate as well as End Product in HMP - Glucose-6-Phosphate.
z Tissues which are never the site of HMP are Non-Lactating Mammary Glands and
Skin.
z HMP activity is low in Skeletal Muscles and Brain (as in Brain, all the Glucose is used
by Glycolysis).
z The high need for Nucleotide Precursors in rapidly dividing cells is provided by
HMP as rapidly dividing cells such as Bone Marrow, Skin , Intestinal Mucosa use
Pentoses to make RNA and DNA.
z Glutathione is a tripeptide (three Amino Acids are Glutamate, Cysteine and
Glycine). It is a Reducing Agent (Reducing Property is due to Sulfhydryl group in
Cysteine).
z Marker for B1 deficiency – Transketolase activity.
z Marker for B2 deficiency – Glutathione Reductase activity.
z Marker for B6 deficiency – Transaminase activity.

z G-6-PD deficiency is the first most common Human Enzyme Deficiency. Pyruvate
Kinase deficiency is the second most common Human Enzyme Deficiency.
z Only RBCs are affected in G6PD deficiency as RBCs lack nucleus and ribosomes. So
they cannot renew the supply of this enzyme. Secondly, HMP is the only means of
generating NADPH in RBCs (in other cells, Malic enzyme is present).
Uronic Acid Pathway

z Uronic Acid Pathway is also a Minor pathway for the Oxidation of Glucose.
z Uronic Acid Pathway synthesizes Glucuronic Acid, Pentoses and Vitamin C.
z Humans cannot synthesize Vitamin C due to deficiency of enzyme : L-Gulonolactone
Oxidase.
z Glucuronate is incorporated into Proteoglycans & it acts as Conjugating Agent (in
Phase II conjugation reactions like Bilirubin Conjugation).
z Essential Pentosuria occurs due to deficiency of Xylitol Dehydrogenase or
Reductase.
z Essential Pentosuria can also occur after consumption of large amounts of fruits that
are rich in Pentoses.
z L-Xylulose appears in urine in Essential Pentosuria which gives positive Benedict’s
Test (but Glucose Oxidase Strip test is negative).
z Garrod’s Tetrad (Pentosuria, Albinism, Alkaptonuria, Cystinuria).
Galactose Metabolism
z Galactose Metabolism, Fructose Metabolism and
Gluconeogenesis uses the enzymes of Glycolysis
so that energy is not wasted to make different
enzymes for many pathways occuring in the cells.
z Source of Galactose is Lactose (milk and milk
products).
z Galactose also obtained by Lysosomal
Degradation of Glycoproteins and Glycolipids.
z Galactose Metabolism occurs exclusively in Liver.
Galactose
ATP
Mg2+ Galactokinase
ADP
Block in
Galactose galactosemia UDPGIc
1-phosphate
Galactose Uridine
1-phosphate NAD+ diphosphogalactose
uridyl transferase 4-epimerase
Glucose
1-phosphate UDPGal
z Galactose Metabolism occurs in 3 steps: Phosphorylation, Synthesis of UDP-

Galactose and Conversion of UDP-Galactose to UDP-Glucose.
z Galactokinase is responsible for the conversion of Galactose to Galactose-1-P.
z GALT (Galactose-1-P Uridyl Transferase) converts Galactose-1-P to UDP-
galactose.
z Conversion of UDP-Galactose to UDP-Glucose is catalyzed by enzyme Epimerase
(UDP Galactose-4-Epimerase or UDP-Hexose-4-Epimerase). This reaction is
reversible.
z UDP-Galactose can be used for the synthesis of Glycolipids, Glycoproteins, GAGs
and Lactose.
z Deficiency of Galactokinase is known as Minor Type Galactosemia. Excess
Galactose gets converted to Galacitol/Dulcitol by enzyme Aldose Reductase.
z Galacitol is hygroscopic in nature and in excess it causes Oil Drop Cataract.
z Deficiency of GALT is known as Classical Type Galactosemia. In this excess
galactose-1-P accumulates in Liver and brain leading to Jaundice and Mental
Retardation, also Oil Drop Cataract occurs.
z Confirmatory Diagnosis for GALT is Direct enzyme assay using Erythrocytes.
z Lactose synthesis in Mammary Glands occurs in Golgi by enzyme Lactose Synthase
(also known as UDP-Galactose: Glucose Galactosyl Transferase) which transfers
Galactose from UDP-Galactose to Glucose.
Fructose Metabolism
z Fructose Metabolism takes place Fructose
mainly in Liver. ATP
z Fructose entry into cells is Insulin Fructokinase
Dependent. ADP
z Fructose ingestion does not lead to Fructose-1-Phosphate

Insulin release. Aldolase B (fructose-1-P
aldolase)
z Fructose is most Lipogenic sugar.
z Fructose gets rapidly metabolized as
DHAP
it bypasses PFK-1 step (rate limiting Glyceraldehyde
step).
ATP
Triose kinase
z Fructose is more sweet than ADP
Glucose.
Glyceraldehyde
z Substrate for Aldolase A and -3-Phosphate
Aldolase C is Fructose 1,6
Enters Glycolysis
Bisphosphate.
z Substrate for Aldolase B is Fructose-1 Phosphate.

z Aldolase A is present in most tissues whereas Aldolase C is present in Brain.
z Energetics of Glucose and Fructose is same. So complete breakdown of Fructose =
32 ATPs.
z Unlike Glucokinase activity, Fructokinase activity is not affected by Insulin.
z Fructose metabolised normally in Diabetic Patient.
z Deficiency of Fructokinase is known as Essential Fructosuria/Benign Fructosuria.
z In Essential Fructosuria, Benedict’s test as well as Seliwanoff’s test is positive.

z Fructose is not raised in blood by Fructokinase deficiency i.e. Fructosemia does not
take place because Hexokinase (a non specific enzyme) can phosphorylate Fructose
as well as other sugars but it has low affinity for Fructose.
z Fructose is excreted in urine because of lack of renal threshold for Fructose.
z Hereditary Fructose Intolerance (HFI) is the deficiency of Aldolase B.
z HFI is an Autosomal Recessive (AR) disease.
z HFI is usually asymptomatic but Fructose ingestion causes symptoms such as
Hypoglycaemia, Jaundice, Lethargy, Hepatomegaly, Vomiting, Seizures and
Irritability.
z HFI is treated by complete exclusion of Fructose, Sorbitol and Sucrose in the diet.
z Sorbitol Pathway synthesizes Fructose in body via Sorbitol.
Osmotic stress
Aldose Sorbitol
reductase dehydrogenase
Glucose Sorbitol Fructose
NADPH NADP+ NAD+ NADH
GSSG GSH
Glutathione
reductase
z Excess Sorbitol in Lens leads to Snow Flake Cataract.

z Sorbitol Pathway leads to decreased NADPH and hence decreased Reduced
Glutathione (antioxidant).
z Sorbitol in excess adds to Diabetic complications.
z Insulin Insensitive Tissues are Peripheral nerves, Renal glomeruli and Lens.
nnn
CHAPTER
ENZYMES 4
ENZYMES
z Enzymes are High Molecular Weight, Highly efficient and specialized Proteins,
which catalyze the Biochemical Reactions.
z Enzymes increase the Rate of the Reaction, without being changed in the overall
process.
z All enzymes are Proteins except
Ribozyme.
z Enzymes are Substrate Specific,
Stereospecific and Thermolabile.
z Active Site is made up of Binding
Sites (usually two) & Catalytic Site.
z Active site (A.S) is Flexible and not
Rigid.
z The A.S of enzyme is formed of Amino Acids which may be placed at long distances
on the Primary Structure, but are brought nearer to each other by 3-D Conformation
of Enzyme.
z Induced Fit Theory – Active Site is flexible, when substrate approaches active site
then there is conformational change which induces the active site to fit the substrate.
z For any Thermodynamically Favourable Reaction, the Free Energy of the Product is
lower than that of Substrate.
z Amount of ΔG tells that a reaction is Thermodynamically Favourable or not.
z Negative ΔG (ΔG<0) → Thermodynamically Stable Reaction.
z Positive ΔG (ΔG<0) → Thermodynamically Unfavourable Reaction (i.e. energy
required).
z ΔG=0 → Reaction is at Equilibrium (i.e. freely reversible).
30 : Enzymes
z Enzyme decreases the Activation Energy as well as Time required by the Reaction.
z Enzyme does not affect Free Energy or Equilibrium of the Reaction.
Activation Energy
z Holoenzyme (Active Enzyme) = Apoenzyme (protein part) + Non-protein part.

z Apoenzyme alone is inactive.
z Non-protein part is of 2 types: Cofactor and Prosthetic Group.
z If the non-protein portion is separable or dialyzable from protein portion, then it is
known as Cofactor.
z If organic, then known as Coenzyme.

z If the Non-Protein Portion is not separable or non dialyzable from Protein.
Portion, then it is known as Prosthetic group.
z Mostly Metal is a Prosthetic Group.
z Enzymes which require Metal Ion as Cofactor are known as Metal Activated
Enzymes.
z Enzymes which require Metal Ion as Prosthetic Group are known as Metallo
Enzymes.
z One third of all Human Enzymes are Metalloenzymes.
z All coenzymes are Cofactors.
z Metals are Cofactors and not coenzymes.
z Coenzyme may be Covalently or Non-Covalently linked.
z Coenzyme Q and Glutathione acts as Coenzyme.
z Some Metalloenzymes contains more than one Metal e.g. Cytochrome Oxidase
contains both Iron and Copper.
Enzymes : 31
z Coenzyme act as a Hydrogen Carrier as well as Carrier of Moieties other than

Hydrogen.
z SAM (S-Adenosyl Methionine) → Methyl donor.
z PAPS (Phospho Adenosyl Phospho Sulphate) → Sulphate donor in synthesis
of GAGs and synthesis of Sulfo Glycosphingolipids and in Steroid Hormone
Catabolism.
z SAM and PAPS are Nucleotides but not Coenzyme.
z Vitamin K is the only Fat Soluble Vitamin which acts as a Coenzyme.
z Lipoic acid is a Coenzyme but not a Vitamin.
z All Water Soluble Vitamins act as Coenzymes.
z All Oxidases need Cu (except Xanthine Oxidase and Sulfite Oxidase which require
Molybdenum).
z All Kinases need Mg++ (But Pyruvate Kinase need K+ >> Mg++).
z All Carboxylases need Mg++.
z Cytoplasmic SOD require Copper but Mitochondrial SOD requires Manganese.
z Isoenzymes are physically distinct form of Enzymes which catalyse same Biochemical
Reaction.
z Isoenzymes may be present in same/different tissues.
z Lactate Dehydrogenase (LDH) is a Tetramer made up of two subunits, H and M.
z LDH-1 (HHHH) is present in Heart.
z LDH-2 (HHHM) is present in Blood (WBC). So known as Functional Plasma
Enzyme.
z In a Normal person, LDH-2 >LDH-1.
z LDH-1 >LDH-2 in Myocardial Infarction (known as Flipped ratio of LDH).
z Creatine Kinase (CK) has two subunits : B (Brain) and M (Muscle).
z CK-1 (BB) is present in Brain.
z CK-2 (MB) is present in Heart.
z CK-3 (MM) is present in Skeletal Muscles.
z LDH-1 and CK-1 has maximum Electrophoretic Mobility.
z LDH-5 and CK-3 has least Electrophoretic Mobility.
z CK-2 is raised in MI after 4-6 hours.
z AST/SGOT is raised in MI after 6-8 hours.
z LDH-1 is raised in MI after 8-10 hours.
z Earliest Marker for MI → Myoglobin but it is Non Specific.
z Last Marker to rise in MI → LDH.
z Most Specific Marker for MI = Troponin I > Troponin T.
z First Marker to fall in MI → Myoglobin.
z Second Marker to fall in MI → CK-MB.
z Last Marker to fall in MI → LDH.
z IMA (Ischemia Modified Albumin) → New Marker for Acute Myocardial Ischemia.
z Isoenzymes of Hexokinase are of four types (Type I, II, III, IV) and Type II being
most abundant and overexpressed in Cancerous Cells.
z Hexokinase IV is known as Glucokinase.
z All Serine Proteases have Catalytic Triad (presence of three Amino Acids at active
site) of Histidine, Serine and Aspartate.
32 : Enzymes
z Examples of Serine Proteases are Chymotrypsin, Trypsin, Elastase, Thrombin,

Plasmin, Complements, Clotting Factors X and XI and PSA (Prostate Specific
Antigen).
z Serine Proteases have role in Tumor Cell Metastasis.
z Chymotrypsin : cleaves Bulky Hydrophobic Amino Acids e.g. Tryptophan, Tyrosine,
Phenylalanine.
z Trypsin : cleaves Basic Amino Acids e.g. Arginine, Lysine.
z Elastase : cleaves small Neutral Amino Acids e.g. Glycine, Alanine.
z Serine Proteases and Aminotransferases have Ping Pong Mechanism of Bi-Bi
Reaction.
z Bi-Bi Reaction has two Substrates and two Products.
z Covalent catalysis often has Ping-Pong Mechanism (first substrate binds and
product released and then the second substrate will bind).
z Oligonucleotide with single base change is used in Site directed Mutagenesis (not
RFLP).
z Single Mutation is detected by RFLP (Restriction Fragment Length Polymorphism).
z Multiple Mutations are directed by Micro Array.
CLASSIFICATION Distinguishing Features

1. Oxidoreductases
Oxidases use O2 as an electron acceptor
Dehydrogenases use molecules other than O2 as electron acceptor (NAD,
FAD, NADP)
Peroxidases use H2O2 as an electron acceptor
Oxygenases incorporate O2 into the substrate
Reductase reduce a substrate by adding Hydrogen (Uses NADPH)
2. Transferases
Methyltransferase transfer one carbon units
Aminotransferases transfer amino groups
Kinases transfer phosphate from ATP
Phosphorylase transfer phosphate from Pi
3. Hydrolases
Phosphatase remove phosphate from a substrate
Any enzyme which break a macromolecule e.g. digestive enzymes
4. Lyases
Synthases link 2 molecules without using ATP
Aldolase produce aldehydes via elimination reactions
Decarboxylase produce CO2 via elimination reactions
Hydratase add or remove water (do not break bond)
Enzymes : 33
5. Isomerases
Racemase interconvert L & D stereoisomers
Mutase transfer groups b/w atoms within a molecule
Epimerase interconvert epimers
6. Ligase
Synthetase link 2 molecules via an ATP-dependent reaction
Carboxylase use CO2 as a substrate
z Oxidoreductase (EC Number 1) carries Oxidation-Reduction Reactions.

z Transferases (EC Number 2) catalyze Transfer of C-, N- or P groups e.g.
Aminotransferase.
z Hydrolases (EC Number 3) uses water to break a bond.
z Lyases (EC Number 4) can break a bond or make a bond, but neither requires water
nor ATP.
z Isomerases (EC Number 5) interconvert Isomers into each other.
z Ligases (EC Number 6) use ATP to make a bond.
z Xanthine Oxidase produce H2O2 but it is not a Peroxidase.
z Oxygenation means O2 is incorporated. In Haemoglobin, there is Oxygenation. If
Oxidation of Hb occurs, then metHb is formed.
z Mutase is Isomerase, not Transferase.
z All Synthases are Lyases.
Exceptions :
1) Nitric Oxide Synthase is an Oxidoreductase
2) Glycogen Synthase and Citrate Synthase → Transferase
3) ATP synthase → Hydrolase
z Oxygenases directly incorporate O2 into the substrate.
z All Hydroxylases are Mono-Oxygenases (Exceptions: Proline Hydroxylase and
Lysyl Hydroxylase are Dioxygenases).
z Mono-Oxygenases are known as Mixed Function Oxidases as they incorporate one
[O] into the Substrate and other [O] into H2 to make H2O.
z Examples of Dioxygenases are Homogentisate Dioxygenase, Tryptophan Pyrrolase.
z Hydroxylase EC Number → 1
z Hydrolase EC Number → 3
z Hydratase EC Number → 4
z Phosphatase is Hydrolase
z Km → that [S] (substrate concentration) at which Velocity of reaction is half of Vmax.
z Km is Michaelis Menton Constant.
z Km is Signature of Enzyme and is inversely proportional to Affinity between Enzyme
and Substrate.
z Km is characteristic of an enzyme and its particular substrate, and reflects the Affinity
of Enzyme for that Substrate.
z Small Km means a High Affinity i.e. low concentration of substrate is needed to
attain half of Vmax.
34 : Enzymes
z Large Km means a Low Affinity i.e. high concentration of substrate is needed to

attain half of Vmax.
z Km does not change with change in [E] or [S]. It is a Constant Value for each enzyme
substrate pair.
z Isoenzymes also have different Km values.
z Km is expressed in same units as [S] i.e. moles/L.
z Graph b/w Velocity and [E] → Linear Graph.
z Graph b/w Velocity and [S] for Simple Enzymes → Rectangular Hyperbola.
z Graph b/w Velocity and [S] for Allosteric enzymes/Regulatory enzymes →
Sigmoidal/S-shape.
z Graph b/w Velocity and Temp → Bell shape.
z Graph b/w Velocity and pH → Bell shape.
z Regulatory/Allosteric Enzymes are those which have extra Regulatory/Allosteric
Site where regulator binds along with the Active Site.
z Allosteric Enzymes are usually Multi Subunit Enzymes.
z Allosteric Enzymes do not follow Michaelis Menton kinetics but follow Hill’s
equation.
z Sigmoidal graph is because of a special property of Allosteric Enzymes i.e.
Cooperativity.
z Cooperative Enzymes are more sensitive in their response to changes in Substrate
Concentrations than other Enzymes.
z Most Rate Limiting Enzymes show Cooperative Kinetics.
z Lineweaver Burk Plot or Double Reciprocal Curve is a modification of Michaelis-
Menton Graph (to convert Michaelis-Menton graph into a straight line graph).
z Enzymes get denatured at extremes of Temperature or pH.
z Hemoglobin Oxygen Dissociation Curve → Sigmoidal/S-shape.
z Myoglobin Oxygen Dissociation Curve → Rectangular Hyperbola.
z A Competitive Inhibitor binds with the Free Enzyme (at Active Site), reversibly,
to form Enzyme-Inhibitor Complex, that is catalytically inactive and cannot bind
Substrate.
z Competitive Inhibitor → Km increase, Vmax same.
z Oxamate is a Competitive inhibitor of Lactate Dehydrogenase.
z Physostigmine is a Competitive inhibitor of Cholinesterase.
z Sulphonamide (Antibiotic) competitively inhibits synthesis of Folic Acid in Bacteria.
z Methotrexate is a Competitive Inhibitor of Dihydro Folate Reductase.
z Dicumarol (Anticoagulant) is a Competitive Inhibitor of Vitamin K Analogue.
z A Non-Competitive Inhibitor binds at the regulatory site and induce a change in
the Active Site so that substrate cannot bind and thus decreases the velocity.
z Non-Competitive Inhibitor → Km and [S] same, Vmax decrease.
z Iodoacetate is a Non-Competitive Inhibitor of Glyceraldehyde-3-P D.
z Heavy metals and Dimercaprol are Non-Competitive Inhibitors of SH group.
z Di-Iso-Propyl Fluorophosphate is a Non-Competitive Inhibitor of Serine Protease.
z Uncompetitive Inhibitor → Both Km and Vmax decrease.
z Example of Uncompetitive Inhibitor is Acetylcholine which inhibits Placental ALP
(Alkaline Phosphatase).
Enzymes : 35
z Suicidal Inhibition is shown by special class of Inhibitors in which enzyme converts

Inhibitor into a Reactive form in its Active Site.
z Examples of Suicidal/Mechanism Based Inhibition: Allopurinol inhibiting Xanthine
Oxidase and Aspirin inhibiting Cycloxygenase.
z Feedback /End Product Inhibition controls the amount of product the cell needs.
z Functional Plasma Enzymes are those enzymes which function in plasma e.g. Blood
Coagulation enzymes.
z Non-Functional Plasma Enzymes are Cell Derived Enzymes which function inside
the cells or tissues e.g. ALT/SGPT.
z Cell damage leads to increased concentration of Non-Functional Plasma Enzymes
in plasma.
z Plasma = Physiologic Fluid.
z Serum = Prepared in lab.
z Serum = Plasma – Clotting Factors.
z Transaminases are used to diagnose Myocardial Infarction (SGOT) and Liver
Diseases (SGPT).
z Alkaline phosphatase is used to diagnose Bone and Liver diseases and
Hyperparathyroidism.
z Prostate Cancer is diagnosed by Acid Phosphatase.
z Therapeutic Enzymes : Streptokinase, Asparaginase, Pepsin, α-1-antitrypsin,
Uricase, Lactase, Trypsin and Chymotrypsin and Collagenase.
z Streptokinase/Urokinase catalyses the Lysis of Intravascular Clots e.g. in MI.
z Asparaginase/Glutaminase used in treatment of Acute Lymphoblastic Leukemia
(ALL).
z Pepsin used in treatment of Chronic Ingestion and Pancreatic Insufficiency.
z α-1-Antitrypsin used in treatment of Emphysema that is caused by deficiency of
α-1-antitrypsin.
z Uricase is used in treatment of Gout.
z Lactase used in treatment of Lactose Intolerance and Penicillin Allergy Treatment.
z Trypsin and Chymotrypsin used for Pain & Inflammation.
z Collagenase used in treatment of Skin Ulcers
z Key Regulatory Reactions or Committed Steps of Metabolic Pathways are often
subjected to Allosteric Regulation.
Allosteric Regulation of Enzyme

When Modulator (M) In the absence of
binds to the regulatory Modulator (M),
subunit, a conformation the substrate can-
change is induced in not binds to the
the catalytic subunit, catalytic subunit of
which enables the enzyme
binding of Substrate
(S) to the active site of
enzyme
z Most common Covalent Modification is Phosphorylation and Dephosphorylation.

36 : Enzymes
z Other Covalent Enzyme Regulation by Covalent Modification

Modifications →
Methylation, Acetylation,
Adenylation, ADP
Ribosylation, Uridylation.
z Phosphate group is
added most commonly
at the -OH group of
Serine (less common at
threonine).
z Di-Isopropyl
Flourophosphate
(DIFP) bind to Serine
residue at position 195 of
Chymotrypsin through
its Phosphate.
I: Phosphorylation, II: Adenylation, III: Uridylylation,

IV: ADP-Ribosylation, V: Methylation
z Enzymes active in Phosphorylated state are Glycogen Phosphorylase, Phosphorylase

kinase, Key enzymes of Gluconeogenesis, HMG CoA Reductase Kinase and ATP
Citrate Lyase.
z Enzymes active in Dephosphorylated state are Glycogen Synthase, Acetyl CoA
Carboxylase, Pyruvate Dehydrogenase, HMG CoA Reductase and PFK-II.
z Compartmentalization of the cells gives a regulation to control the enzymatic activity.
z Enzyme synthesis can be induced or repressed at the level of genes.
z House Keeping or Constitutive Genes are those genes which are always active E.g.
Enzymes of TCA cycle.
z Inducible Genes are the genes which can be activated whenever required E.g.
Enzymes of Gluconeogenesis.
z Ubiquitin Proteosome Pathway (UPP) is ATP Dependent Pathway for degradation
of proteins/ enzymes.
z UPP occurs in Cytoplasm and Nucleus and is for damaged or short lived proteins.
z Ubiquitin protein is highly conserved, small, globular, non enzyme protein and is
recycled.
z ε-amino of Lysine in protein is attached to C-teminal Glycine of Ubiquitin.
z ATP Independent Proteolytic Pathway occurs in Lysosomes and Enzyme used is
Hydrolase.
z Proteins having Amino Acids such as Arginine and Acetylated Alanine at
N-terminal ends have short half lives and are rapidly degraded.
z Proteins rich in Proline, Glutamate, Serine And Threonine (PEST sequence) are also
degraded.
z Zymogens are inactive precursors of enzymes.
nnn
CHAPTER
AMINO ACIDS 5
AMINO ACIDS – CLASSIFICATION & METABOLISM
z Amino group (-NH2) is always on left side and Acid group (-COOH) is always on
right side in an Amino Acid.
z Any compound having Asymmetric Carbon shows both Optical and Structural
Isomerism.
z All Amino Acids have 1 Asymmetric Carbon except - Glycine, Threonine and
Isoleucine.
z Glycine has no asymmetric carbon.
z Threonine & Isoleucine have 2 asymmetric carbons.
OH NH2 CH3 NH2

CH3 CH C COOH CH3 CH2 CH C COOH
H H
threonine isoleucine
z Amino Acids ionize to give negative charge on Carboxy group and positive charge
on Amino group and the Net Charge is zero, hence known as Zwitter-Ion or
Ampholyte.
z pI is Isoelectric pH → that pH at which Zwitter ions exist.
z At pI (Isoelectric pH), a protein will precipitate.
basic acidic
group H group H

H2N C COOH +H N C COO
3
R R
amino acid zwitterion
z All Amino Acids are L-α-Amino Acids, which are present in Proteins. But in body,
Free Amino Acids are present (i.e. those which are not in proteins), which can be L
or D or α/β/Y.
z Amino Acid abundant in Protein → L-form.
z Amino Acid abundant in Body → L-form.
z Amino Acid present in Protein → always ‘L’ form and always α.
z Amino Acid present in Body → Both ‘L’ and ‘D’ forms.
38 : Amino Acids
z Free Amino Acids may be L-form or D-form.

z Free Amino Acids may be α/β/Y.
z Essential Amino Acids are those that cannot be synthesized in our body, hence are
required in diet.
z Non-Essential Amino Acids are those that can be synthesized in our body, hence
are not required in diet.
z Semi-Essential Amino Acids are those that can be synthesized in our body but to
some extent.
z A total of 20 Amino Acids are required in body for synthesizing Proteins of which
8 are Essential.
z 21st and 22nd Amino Acid are Selenocysteine (given by codon UGA) and Pyrrolysine
( given by codon UAG) respectively. These two Amino Acids are formed by
modification of Stop Codons which is a Co-Translational Modification.
z 22 Amino Acids are encoded by DNA i.e. they are not formed by Post Translational
Modifications.
Pyrrolysine
O NH2
X OH
N
H
N O
Lysine NH2
OH
H2N
z Precursor Amino Acid for Pyrrolysine is Lysine.

z Pyrrolysine is not found in humans.
z Precursor Amino Acid for Selenocysteine is Serine.
OH SH SeH
H H H
O O O
H2N H2N H2N
OH OH OH
Serine (Ser) Cysteine (Cys) Selenocysteine (Sec)
z Aromatic Amino Acid with basic properties is Histidine.

z Aromatic Amino Acid with –OH group is Tyrosine.
z Arginine and Histidine are Semi-Essential Amino Acids (mark Arginine if both
given as Arginine is more towards Essential category as compared to Histidine).
Amino Acids : 39
Arginine Histidine
O O
H2N CH C OH H2N CH C OH
CH2 CH2
CH2
N
CH2
NH
NH
C NH
NH2
z Histidine can be formed in Adults but not in Children.

z Amino Acid responsible for Flexibility of Proteins is Glycine (because of its smallest
side chain).
z Glycine is not found in α-helix, but found H
in β-turns/bends.
H2N
z Glycine is the simplest, smallest, Non- C COOH
Essential, Non-Polar, and Glucogenic H

Amino Acid. glycine
z Glycine can be synthesized from:

1) CO2 and NH4+ by Glycogen Synthase (N5, N10 Methylene THF involved)
2) From Glyoxylate by Glycine Amino Transferase/ Glycine Transaminase (PLP
required).
3) From Serine by Serine Hydroxyl Methyl Transferase (reversible reaction) – PLP
and Folic Acid required (N5, N10 Methylene THF involved).
4) From Threonine by Threonine Aldolase.
z Glycine Cleavage System occurs in Mitochondria and is a Major Pathway for
Glycine Degradation.
z Primary Hyperoxaluria is defect in Glycine Transaminase associated with Impaired
Glyoxylate to Formate conversion, which diverts excess of Glyoxylate to form
Oxalate which can form Oxalate Stone in Kidneys.
z Oxalate in urine can appear in B6 deficiency.
z Alanine (Non-Polar) is the most Glucogenic CH3
Amino Acid (3C). C COOH
H2N
H
Alanine
z All branched chain Amino Acids are Essential and Non-Polar.

z Isoleucine is the most Non-Polar Amino Acid and is both Glucogenic and Ketogenic.
40 : Amino Acids
z Amino Acid that can act as a fuel for Brain is Isoleucine.

z Leucine is purely Ketogenic.
z Valine is Glucogenic.
- - -
COO COO COO
+ + +
H3N C H H3N C H H3N C H
CH CH H C CH3
H3C CH3 CH CH2
H3C CH3 CH3
Valine Leucine Isoleucine
z Maple Syrup Urine Disease (MSUD) is Autosomal Recessive disease in which there
is a defect in the Oxidative Decarboxylation step (in the catabolism of Branched
Chain Amino Acids).
z Enzyme defect in MSUD is branched chain α-Ketoacid Dehydrogenase/ Branched
Chain α-Keto Acid Decarboxylase (Multi enzyme complex).
z Clinical features of MSUD are Burnt Sugar like Odour Of Urine, Ketosis, Vomiting,
Feeding Problems, Mental Retardation, Abnormal Muscle Tone
z If MSUD is not treated , then Coma or Death may occur.
z Patients with rare Thiamine Dependent Variant of MSUD give response when large
doses of Vitamin B1 is given.
z Isovaleric Acidemia – Defect in FAD Dependent Dehydrogenase (Isovaleryl CoA
Dehydrogenase) which specifically degrades Isovaleric Acid (involved in Metabolism
of Leucine) – Sweet feet odour of urine.
z All Aliphatic Amino Acids (Valine, Isoleucine, Leucine, Alanine, Glycine) are Non-
Polar.
z Least Non-Polar is Glycine.
z Glycine Metabolism is linked with THF & Vitamin B6.
z Primary Hyperoxaluria is defect in enzyme Glycine Transaminase.
z Causes of Secondary Hyperoxaluria are Vitamin B6 deficiency, Vitamin C Toxicity
& Ethylene Glycol Poisoning.
z In Non-Ketotic Hyperglycinemia (or Glycine Encephalopathy), there is defect in
Glycine Cleavage System.
z In Glycinuria, there is defective reabsorption of Glycine due to defective transporter
for Glycine & Proline.
z Uses of Glycine → Formation of Haem, Glutathione, Serine, Creatine, Creatinine,
Choline, Betaine and Conjugation of Primary Bile Acids.
z Creatine is formed from Arginine & Glycine and SAM acts as a Methyl Donor.
z Choline and Betaine Metabolism is interrelated with Folate metabolism.
Amino Acids : 41
z Aromatic Amino Acids: Phenylalanine, Tyrosine, Tryptophan.
z Phenylalanine is converted to Tyrosine by enzyme Phenylalanine Hydroxylase

(mono-oxygenase), Tetrahydrobiopterin (THB) & NADPH is required as Coenzyme.
z Catecholamines are Dopamine, Epinephrine & Nor-Epinephrine.
z First Catecholamine to be synthesized is Dopamine.
z Catecholamine with Methyl Group is Epinephrine.
Synthesis of catecholamines (Dopamine, NE and E) from tyrosine
z Epinephrine formation occurs in Periphery (not in CNS), 80 % of it occurs in

Adrenal Medulla.
z Catabolic end product of Epinephrine & Nor- Epinephrine is VMA (Vanillyl
Mandelic Acid).
z Rate Limiting Enzyme in Catecholamine Synthesis is Tyrosine Hydroxylase
z Precursor of all three Catecholamines are L-DOPA (Di-Hydroxy Phenyl Alanine).
z Catabolic End Product of Dopamine is Homovanillic acid.
42 : Amino Acids
z Normal VMA levels are 2-6 mg/ 24 hours.

z VMA levels are increased in Pheochromocytoma & Neuroblastoma of Adrenal
Glands.
z THB also required for Tyrosine to DOPA conversion, Tryptophan to 5-OH
Tryptophan conversion and Arginine to Citrulline conversion.
z THB is required mainly for Hydroxylases.
z Phenylalanine, Tryptophan, Tyrosine are both Glucogenic and Ketogenic.
z Phenylketonuria (PKU) is due to defect in enzyme Phenylalanine Hydroxylase.
z Phenylketonuria patient is normal at birth.
Biochemistry of Phenylketonuria
z Clinical features of Phenyl- Phenyl ketonuria

ketonuria are Mousy/Musty odour
(due to Phenylacetate), Severe z Mousy odor
Mental Retardation, microcephaly, z Hypopigmen-
exaggerated tendon reflex, seizures, tation
hyperactivity, rash, wide spaced z Seizures
teeth, Hypopigmentation (Melanin

not formed because of Tyrosine
deficiency)- fair skin, blue eyes. z Severe mental
z Screening of Phenylketonuria is done by retardation
Tandem Mass Spectrometry (accurate,
precise, low false positive results).
z Gutherie’s Test (Bacterial Inhibition Assay) for Phenylketonuria is now replaced by

Tandem Mass Spectrometry.
z Hyperphenylalaninemia can also occur from THB deficiency.
z Tyrosine is used for the synthesis of Catecholamines, Thyroid Hormone and the
pigment Melanin.
Amino Acids : 43
z Albinism (milky white skin, white hair, red eye color) is absence of pigment
Melanin from skin, hair and eyes. Enzyme deficient is Tyrosinase (an Oxidase and
Cu required).
z Oculo Cutaneous Albinism associated with defects in Vision and Photophobia.
z In Vitiligo, enzyme Tyrosinase is normal but there is lack of Melanoblasts in
regional areas.
z Alkaptonuria is associated with defects in Catabolism of Tyrosine and
Phenylalanine.
z Alkaptonuria (Autosomal Recessive) is part of Garrod’s Tetrad.
z Benedict’s Test is positive in Alkaptonuria due to Homogentisic Acid which is
Reducing in nature.
z Fresh urine of Alkaptonuria patients is normal color.
z But urine on long standing turns black due to oxidation of Homogentisic Acid.
z There is no Mental Retardation in Alkaptonuria.
z Polymerization of Homogentisic Acid forms Alkapton Bodies which gets
accumulated in Cartilages giving Bluish Black Connective Tissues.
z Enzyme deficient in Alkaptonuria is Homogentisate Dioxygenase.
z Characteristics of Alkaptonuria are Dark staining of Diapers, Deposition of Black
Pigments in joints, Cartilage and Collagenous Tissue (Ochronosis)
z Nitisinone is used in treatment of Alkaptonuria.
z Nitisinone inhibits PHPP (Para Hydroxy Phenyl Pyruvate) Hydroxylase.
z Glutathione is required in the Catabolism of Phenylalanine & Tyrosine.
z Homogentisic Acid is an intermediate in the Catabolism of Tyrosine.
z Tyrosinemia Type I or Tyrosinosis is most common Tyrosinemia– defective
Fumaryl Aceto Acetate Hydrolase – Terminal Enzyme in Tyrosine Catabolism –
characterized by Boiled Cabbage/Rancid Butter like Odour of Urine, also known as
Hereditary Tyrosinemia or Hepato-Renal Tyrosinemia.
z Tyrosinemia Type II is known as Richner Hanhart Syndrome or Oculo-Cutaneous
Tyrosinemia is deficiency of Tyrosine Transaminase.
z Tyrosinemia Type III is Neonatal Tyrosinemia- deficiency of PHPP (Para Hydroxy
Phenyl Pyruvate) Hydroxylase.
z Tryptophan Pyrrolase is a Dioxygenase and contains haem.
z Uses of Tryptophan – Formation of Serotonin, Melatonin & Niacin.
z Rate Limiting Enzyme in Niacin synthesis is QPRTase (Quinolate Phospho Ribosyl
Transferase).
z 60 mg Tryptophan forms 1 mg Niacin.
z Serotonin is synthesized in Intestine & Placenta and is a Neurotransmitter
responsible for Mood Elevation and Temperature Regulation.
z Melatonin is Acetyl Methyl Serotonin.
z Melatonin is synthesized in Pineal Glands and is the Neurotransmitter responsible
for Circadian Rhythm.
z Excretory product of Serotonin is 5-HIAA (Hydroxyl Indole Acetic Acid).
z Tryptophan forms 5-OH Tryptophan which is converted to Serotonin (5-OH
Tryptamine) and then Melatonin.
44 : Amino Acids
z Tryptophan also forms Niacin/ Vitamin B3 (known as Atypical vitamin).

z Atypical Vitamins are Vitamin B3 & Vitamin D.
z Vitamin B-6/ PLP deficiency leads to increased Xanthurenic Acid.
z Hartnup’s Disease – Autosomal Recessive, Rare Disorder – Defect in Neutral
Amino Acid Transporter – Failure to absorb Tryptophan from Intestine and also
reabsorb it from Kidneys.
z Massive Aminoaciduria (mainly Tryptophan) without a corresponding increase in
Plasma Amino Acid level is characteristic of Hartnup’s Disease.
z Mutation of Hartnup’s Disease is on gene SLC6A 19.
z In Carcinoid Syndrome, Tryptophan used to form excess Serotonin, so Tryptophan
to Niacin conversion not occurring, so Pellagra occurs.
z Catabolic end product of Histidine is Glutamate formed via FIGLU.
z Histidine has maximum Buffering Capacity because Histidine is the only Amino
Acid which can ionize within the physiological pH range.
z Arginine > Lysine > Histidine (in terms of Polarity).
z All Basic Amino Acids (Histidine, Arginine, Lysine) and Acidic Amino Acids(
Aspartic Acid, Glutamic Acid) are Polar.
z Arginine is Glucogenic (Catabolic End Product – α-Ketoglutarate).
z Asparagine synthesis require ATP and Glutamine is –NH2 donor.
z All Acidic Amino Acids and their Amides are Glucogenic.
COO COO COO COO

+ +
+ +
H3N C H H3N C H H3N C H H3N C H
CH2 CH2 CH2 CH2
CH2 C CH2
COO
H2N O
C
COO
H2N O
Aspartate [D] Glutamate [E] Asparagine [N] Glutamine [Q]

(Asp) (Glu) (Asn) (Gln)
z Asparagine is the site for N-glycosidic bonds.

z Serine and Threonine are –OH containing Amino Acids. Also the site for
O-glycosidic bonds.
NH2 OH NH2
HO CH2 CH COOH CH3 CH CH COOH

Serine Threonine
Amino Acids : 45
z Cysteine and Methionine are Sulphur containing Amino Acids.
Cysteine Methionine
z Cysteine is oxidised to Cystine (Disulphide bond formation).

z Methionine forms SAM (S-Adenosyl Methionine)- Methyl donor.
z MAT (Methionine Adenosyl Transferase) converts Methionine to SAM (S-Adenosyl

Methionine).
z MAT 1 & 3 is in Liver.
z MAT-2 is in Extra-Hepatic Tissues.
z SAM and Coenzyme A are High Energy Compounds but they don’t have Phosphate.
z Cysteine is not an Essential Amino Acid as long as Methionine is available in diet.
z Tyrosine is not an Essential Amino Acid as long as Phenylalanine is available in diet.
z Cystine is made up of two molecules of Cysteine.
z Cysteine has –SH group but Cystine has disulfide bond.
46 : Amino Acids
z Homocystine is made up of two molecules of Homocysteine.

z Homocysteine is increased in Blood in vitamin B6, B9 or B12 deficiency. In urine,
Homocystine is increased.
z In Type I Homocystinuria, Cysteine becomes Essential.
z In Type II Homocystinuria, Cysteine can be synthesized in body.
z For the formation of Cysteine, two Amino Acids are required i.e. Methionine &
Serine and one vitamin required is Vitamin B6.
z Cyanide Nitroprusside Test is positive for Homocystinuria, Cystinuria & Cystinosis.
z In Cystinuria, there is a defect in Dibasic Amino Acid Transporter.
z In Cystinosis, there is defect in Cystine Transporter (Cystinosin) in Lysosomes, so
it is a generalized Lysosomal Storage Disorder.
z Cystinuria → Most common genetic error in Amino Acid transport - ‘COAL’ carrier
defective - 4 Amino Acids: Cysteine, Ornithine, Arginine, Lysine appear in urine.
z All Amino Acids have Primary Amino Group
except Proline having Secondary Amino Group.
z Proline (Imino group) can never be
accommodated in α-helix.
z Catabolic end product of Proline is Glutamate.
So Glucogenic: Proline
Wheat lacks Lysine
Pulses lack Methionine
AMINO ACID CATABOLISM & UREA CYCLE

z Transamination is the transfer of NH2 group from Amino Acid to corresponding
α-Keto acid.
z Transamination reactions are reversible and require PLP (derivative of Vitamin B6)
as coenzyme.
z Removal of Amino Group from Amino Acids form α-Keto acids.
z Transamination is the first reaction in the Catabolism of Amino Acids.
z On addition of –NH2 group, α-Ketoglutarate forms Glutamate by SGOT/SGPT and
further forms Glutamine by enzyme Glutamine Synthetase (requires ATP).
z On addition of –NH2 group, Oxaloacetate forms Aspartate and further forms
Asparagine.
z Glutamate is regarded as collector of Amino Groups.
z Glutamate is the only Amino Acid that can undergo Oxidative Deamination.
z Transdeamination is coupling of Transamination (occurs in all organs) & Oxidative
Deamination (occurs in liver).
z Only α-Amino group of Amino Acids takes part in Transamination. One exception
is that δ-Amino group of Ornithine takes part in Transamination and synthesize
Proline.
z SGOT: Serum Glutamate Oxaloacetate Transaminase – also known as AST
(Aspartate Transaminase)- Aspartate loses amino group and forms OAA and
α-Ketoglutarate takes amino group and forms Glutamate.
Amino Acids : 47
z SGPT: Serum Glutamate Pyruvate Transaminase – also known as ALT (Alanine

Transaminase)- Alanine loses amino group and forms Pyruvate and α-Keto
Glutarate takes amino group and forms Glutamate.
z Glutamine Synthetase converts Glutamate to Glutamine (ATP used) and
Glutaminase converts Glutamine to Glutamate (no ATP used).
z Glutamine is a Non-Toxic Storage and Transport Form of Ammonia in Blood.
z GDH (Glutamine dehydrogenase) helps in Oxidative Deamination of Glutamate
to release Ammonia in Liver for the entry into Urea Cycle. This enzyme uses either
NAD or NADP.
z Glutamate forms GABA (Gamma Amino Butyric Acid).
z Actiavtors of GDH : NAD, ADP & GDP.
z Inhibitors of GDH : NADH, ATP & GTP.
z Major transporter of NH3 from Body & Brain is Glutamine and from Muscles it is
Alanine.
z Amino Acids which cannot form Glutamate and cannot undergo transamination are
Proline, Hydroxy- Proline, Lysine and Threonine.
z Glucose – Alanine cycle is also known as Cahill cycle – Transport of ammonia from
Muscles to Liver – occurs in Fasting Muscles.
z Excess Ammonia increases Glutamine and decreases α-Ketoglutarate and Glutamate

and BUN (Blood Urea Nitrogen) is also decreased.
z Clinical features of Hyperammonemia are Lethargy, Mental Confusion, Agitation,
Blurred Vision, Slurred Speech, Cerebral Edema, Vomiting, Fine Tremors,
Hyperventilation and if not treated then.
z Ammonia Encephalopathy – Increased Glutamine is osmotically active and leads to
cell swelling, specially Brain cells – Symptoms are Cerebral Oedema and Vomiting.
48 : Amino Acids
z Urea Cycle occurs in Liver.

z Urea Cycle occurs in both Mitochondria and Cytoplasm.
z Rate Limiting Enzyme of Urea Cycle–Carbomoyl Phosphate Synthetase–I (CPS-I).

z Arginine is the immediate precursor of Urea.
z Urea is synthesized by Arginase.
z Aspartate is transported from Mitochondria to Cytoplasm by Citrin Transporter.
z Ornithine and Citrulline Antiport is present in IMM, which transports
Citrulline from Mitochondria to Cytoplasm and Ornithine is transported into the
Mitochondrial Matrix.
z NAG Synthase is regarded as 6th enzyme of Urea Cycle, which synthesize NAG
(N-Acetyl glutamate). NAG is the Allosteric Activator of CPS-I (Rate Limiting
enzyme of Urea Cycle).
z NAG Synthase is activated by Glutamate & Arginine and it is inhibited by N-Acetyl
glutamate.
z 4 ATPs are used to synthesize one molecule of Urea.
z Ornithine has a catalytic role in Urea Cycle.
z Carbon of urea is derived from CO2 or bicarbonate (HCO3-), Two nitrogens are
derived from aspartate and ammonia.
z Urea Cycle Enzyme deficient in Brain: OTC (Ornithine Trans Carbamoyalase).
z Urea Cycle Enzyme deficient in Kidneys : Arginase.
z End product of Urea Cycle in Kidneys: Arginine.
z Major source of Arginine (a Semi-Essential Amino Acid) is Kidneys.
z Main Clinical Features of Urea Cycle Disorders are Hyperammonemia,
Encephalopathy And Respiratory Alkalosis.
z OTC (Ornithine Transcarbamylase) Deficiency (Orotic Acid, OMP, UMP increased)
is the most common Urea Cycle Disorder. It is known as Hyperammonemia Type-II.
z Hyperammonemia Type I is deficiency of CPS-I.
Amino Acids : 49
z Hyperammonemia Type I & Hyperammonemia Type II are most severe Urea Cycle
defects as ammonia is present in inorganic form which is highly toxic.
z Arginine is the first line treatment of Urea Cycle Disorders but it is contraindicated
in Hyperargininemia.
z Sodium Benzoate and Phenylbutyrate are Ammonia Scavenging Agents.
z Deficiency of enzyme Arginosuccinate synthetase leads to Citrullinemia Type I.
z Defect in Citrin Transporter (transports aspartate) leads to Citrullinemia Type II.
z Phenyl Butyrate is more effective than Sodium Benzoate in treating Urea Cycle
disorders.
z Increased Gluconeogenesis from Amino Acids increase Urea formation.
PROTEINS – BONDS & STRUCTURES
z Proteins are polymers of Amino Acids

z Amino Acids are joined by strong Covalent
Amide/ Peptide bonds.
z If Amide bond is present in proteins then it is
called Peptide bond.
Protein foods
z Peptide bond → rigid and planar →

partial double bond character → “trans”
configuration.
z Unsaturated Fatty Acid Double Bond → “cis”.

z If < 50 Amino Acids in a chain → known as Peptide.
z If > 50 Amino Acids in a chain, then known as Polypeptide or Protein.
z Protein digestion begins in Stomach. HCl from parietal cells just denature the
Proteins and activates Pepsinogen to Pepsin.
z Pepsin is an Endopeptidase.
z Pancreatic Enzymes are Trypsin, Chymotrypsin, Elastase and Carboxypeptidases
A+ B.
z Endopeptidases cut at the Carboxy Terminal of Specific Amino Acids.
z Exopeptidases cut at the C- terminal of Protein.
z Carboxypeptidases A: Preferably acts on Alanine and Branched Chain Amino Acids
(BCAA).
z Carboxypeptidases B: Preferably acts on Basic Amino Acids e.g. Arginine, Lysine.
z Small Intestinal Enzymes are Aminopeptidases, Dipeptidases and Tripeptidases
(Also secreted as Zymogens).
z Aminopeptidases: An Exopeptidase, cut peptide bond at N-terminal of Protein.
50 : Amino Acids
z Dipeptidases and Tripeptidases: Cleaves Di and Tri peptides into their individual
Amino Acids, which are absorbed.
z Primary Structure: Sequence of Amino Acids joined by Peptide bonds.
z Secondary structure: Folding of Primary Structure e.g. α-helix, β-sheet, β-turn/
bends, α-chain in Collagen.
z Each turn of α-helix contains 3.6 Amino Acids.
z α-helix is Rigid and Right handed - Only Interchain Hydrogen bonds present.
z β-sheet has both Interchain and Intrachain Hydrogen bonds – fully extended chain
– may be parallel or antiparallel.
z Tertiary Structure: Single fully folded Functional Polypeptide chain.
z Quaternary Structure: More than one fully folded Functional Polypeptide chains.
z Monomeric proteins do not contain Quaternary structure.

z Proline is never found in α-helix.
z Proline and Glycine are found in beta turns.
z Amino Acid with Bulky R groups : Tryptophan.
Amino Acids : 51
z Amino Acid with Branch Chain at beta carbon : Valine.

z Mass Spectroscopy is the best technique to determine the Primary Structure of
Proteins.
z X-ray crystallography is the best technique to detect Secondary, Tertiary or
Quaternary Structure of Proteins.
z NMR Spectrometry is the best technique to detect Secondary, Tertiary or Quaternary
Structure of Non-Crystallizable Proteins or Membrane Proteins
z X-ray crystallography detects 3-D Structure of any Macromolecule.
z Most powerful technique for screening inborn errors of metabolism is Tandem
Mass Spectrometry.
z Tandem Mass Spectrometry, also known as MS/MS or MS2 → in the first stage of
MS, precursor (characteristic of given analyte) is selected, then fragmentation occurs
by collision induced dissociation to produce ions. Then in second stage of MS, ions
are separated according to mass to charge ratio, and then detected. The fragments
then reveal aspects of the chemical structure of the precursor ion.
z Denaturation of Proteins is sometimes reversible. Denaturing Agents are Heat,
Urea, Guanidine, UV rays, IR rays, Strong Acids or Alkalis.
z Coagulation is clumping of Denatured Protein and is always irreversible.
nnn
CHAPTER
LIPIDS 6
LIPID BIOCHEMISTRY
z Fatty Acid consists of a Hydrophobic Hydrocarbon Chain (Non-Polar) and a
Hydrophilic Carboxyl Group (Polar) which is ionized at pH 7. In case of Long Chain
Fatty Acids, the Non Polar Portion is predominant. In case of Short Chain Fatty
Acids, the Polar Portion is predominant.
z Terminal Methyl Group in a Fatty Acid is called ω-carbon (omega).
z C2 in case of Fatty Acid is called
α-carbon (alpha).
β-carbon (beta).
g-carbon (gamma).
z Acid + Alcohol = Ester bond.
z Fatty Acid (Polar) + Alcohol (Polar) = Fat (Non-polar).
z Acid + Amino = Amide bond.
z Simple Lipids are only made up of 2 components i.e. Fatty Acid (FA) and Alcohol.
z Complex Lipids are made up of three components → Fatty Acid, Alcohol and Other
Component (Phosphate or Carbohydrate) e.g. Phospholipids & Glycolipids.
z Alcohol in Simple Lipids is Glycerol (3C).
z Mono-Acyl Glycerol (MAG) : One Fatty Acid + Glycerol.
z Di- Acyl Glycerol (DAG) : Two Fatty Acids + Glycerol.
z Tri- Acyl Glycerol/Neutral Fat/Triglycerides (TGs) : Three FAs + Glycerol
z Mono Acyl Glycerol & Di Acyl Glycerol is Amphipathic. But Tri Acyl Glycerol is
Non Polar.
z TGs are the main lipids present in diet and also the main Storage Form of Lipids in
the body (in Adipose Tissues).
z Hormone Sensitive Lipase present in Adipose Tissues breaks down TGs. It is active
in Fasting state.
z Phospholipids/ Phosphoglycerides = Alcohol +
Fatty Acid + Phosphate.
z Phospholipids are of two types: Glycero-
Phospholipids and Sphingo-Phospholipids.
z Parent alcohol is Glycerol (3 C) in Glycero-
Phospholipids.
54 : Lipids
z Parent alcohol is Sphingosine (20 C) in Sphingo-Phospholipids.

z Phosphatic Acid = Glycerol +2 Fatty Acids +Phosphate.
z Phosphatidic Acid is the simplest Phospholipid and is also the precursor of all
Phospholipids.
z Lecithin is Phosphatidyl Choline (Glycerol + 2 Fatty Acids + Phosphate + Choline).
z Most abundant Phospholipid in HDL is Lecithin.
z Cephalin is Phosphatidyl Ethanolamine (Glycerol + 2 Fatty Acids + Phosphate +
Ethanolamine).
z Lecithin and Cephalin are the most abundant Phospholipids in most eukaryotic
cells.
z Phosphatidyl Serine is involved in Apoptosis.
z SAM (S-Adenosyl Methionine) is used in the synthesis of Lecithin from Cephalin.
z Lecithin is the largest body store of Choline and most abundant Phospholipid of
Cell Membrane.
z L/S ratio (Lecithin : Sphingomyelin ratio) > 2 indicate Fetal Lung Maturity, which
occurs at 32 weeks of gestation.
z Dipalmitoyl Lecithin is used as a Surfactant in Lungs.
z Cephalin is present Brain/CNS.
z Phospholipases are the enzymes which break down Phospholipids. These are of four
types. Phospholipase A1 breaks the first FA i.e. from C1. Phospholipase A2 breaks
the FA from C2. Phospholipase C breaks Phosphoryl base from rest of the Molecule.
Phospholipase D acts between Phosphate and the base.
z Cardiolipin/ Phosphatidyl Glycerol = Glycerol + 2 Phosphatidic Acids.
z A Phospholipid which can be Antigenic is Cardiolipin (present in Inner
Mitochondrial Membrane).
z Barth Syndrome – a rare X-linked Disorder, occurs due to defect in Cardiolipin
Modelling. Patients have Muscle Weakness, Neutropenia And Cardiomyopathy.
z Platelet Activating Factor (PAF) is a Glycero-Phospholipid in which C-1 of
Glycerol is attached to Saturated Alkyl group with an Ether Linkage and C-2 is
having an Acetyl Residue (not Fatty Acid). It activates Inflammation, Thrombosis
and aggregate Platelets. PAF lowers Blood Pressure and is one of the most potent
Bioactive Molecules known (effective at low concentrations).
z Sphingosine is a 20-C Amino Alcohol, which is synthesized from Serine and
Palmitate.
z Ceramide = Sphingosine + FA (mostly Long Chain FA).
z Ester bond present usually in all Fats, but Ceramide is a lipid having Amide bond.
z Sphingomyelin = Sphingosine + FA + Phosphate + Choline.
z For Sugar Activation, UDP (Uridine Diphosphate) is used in Carbohydrate
Synthesis. Similarly, in Phospholipid Synthesis, CDP (Cytidine Diphosphate is
used).
z Glycolipids or Glycosphingolipids are the source of ABO Blood Group Antigens,
where Carbohydrate is the Antigenic Determinant.
z Glycolipid/Cerebrosides = Alcohol + Fatty Acid + Carbohydrate (Glucose/
Galactose).
z In Glycolipids, always Sphingosine Alcohol is present (not Glycerol). Hence also
known as Glycosphingolipids.
Lipids : 55
z If Glucose is added to Ceramide, then it is known as Gluco-cerebroside or Glucosyl-

Ceramide.
z If Galactose is added to Ceramide, then it is known as Galacto-cerebroside or
Galactosyl-Ceramide.
z Gluco-cerebrosides are never found in CNS but Galacto-cerebrosides are always
found in CNS.
z If Oligosaccharide Chain containing Sialic Acid/N-Acetyl Neuraminic Acid
(Polar Component) is further added to Cerebrosides, then known as Gangliosides
(Complex Glycolipids).
z Gangliosides are found in Ganglion Cells of CNS and are never found in Liver.
z Simplest Ganglioside is GM3 Ganglioside.
z GM1 acts as receptor for Cholera Toxin in Intestine.
z If Sphingomyelin accumulates, then it is Neimann’s Pick disease. Enzyme deficiency
is Sphingomyelinase which is a kind of Phospholipase C.
z If Glucocerebrosides accumulates then it is Gaucher’s Disease. Enzyme deficiency
is Glucocerebrosidase.
z There is no Mental Retardation in Gaucher’s Disease.
z Gaucher’s disease is the most common Lysosomal Storage Disease.
z β-Galactosyl Ceramidase deficiency leads to Krabbe’s disease.
z Fabry’s disease (α-Galactosyl Ceramidase deficiency) is XR (X-Linked recessive).
z If Gangliosides accumulate then it is Tay Sach’s Disease . Enzyme deficiency is
Hexosaminidase A.
z There is no Hepatosplenomegaly in Tay Sach’s disease.
z Sphingolipidoses are Niemann’s Pick Disease, Gaucher’s Disease, Tay Sach’s Disease,
Fabry’s Disease, Krabbe’s Disease, Farber’s Disease, generalized Gangliosidosis,
Metachromatic Leukodystrophy, Sandhoff’s Disease.
z Sphingolipidoses with no Mental Retardation is Gaucher’s Disease, Fabry’s Disease.
z Sphingolipidoses with no cherry red spots is Gaucher’s Disease, Fabry’s Disease.
z VLCFA (Very Long Chain Fatty Acids) (> 22C) are usually present in Brain.
z Coconut and Coconut Oil has highest content of Medium Chain Fatty Acids.
z Olive Oil has Long Chain (C16-18) Unsaturated FA.
z Butter has Long Chain (C16-18) Saturated FA.
z Beef Fat has Long Chain ( > C18) Saturated FA.
z Number of Hydrogens in case of a Saturated Fatty Acid (no double bond) are double
the number of carbons e.g. Acetic acid (2C) has four Hydrogens.
z Usually unsaturated FAs have Double Bonds in ‘cis’ configuration but on prolonged
heating, it gets converted to ‘trans’ configuration.
z Trans Fats are dangerous as they increase TG, LDL and decrease HDL i.e. increase
the risk of Cardiovascular Diseases and also Body’s Inflammatory Response.
z Monounsaturated Fatty Acids (MUFAs): one Double bond present e.g. Oleic acid
(18C, one double bond).
z Polyunsaturated Fatty Acids (PUFAs) are known as Essential FAs as they are
essential in diet. PUFAs are divided into ω-3 (omega 3) and ω-6 (omega 6) categories.
z ω-3 Essential FAs are Cervonic Acid, α-Linolenic (18 C, 3 Double bonds) acid and
Timnodonic Acid (20 C, 5 Double bonds).
56 : Lipids
z ω-6 Essential Fatty Acids are Gamma-Linolenic Acid (18 C, 3 Double bonds),
Linoleic acid (18 C, 2 Double bonds), Arachidonic Acid (20 C, 4 Double bonds).
z Most essential Fatty Acid is Linoleic Acid.
z α-Linolenic Acid is the precursor of ω-3 category.
z Linoleic Acid is the precursor of ω-6 category.
z Cervonic Acid / Docosa Hexaenoic Acid (DHA) has 22C and 6 double bonds.
DHA is important for Brain development in Children and decreased amount leads
to increased risk of Retinitis Pigmentosa. DHA is present in high concentration in
Sperm, Retina, Cerebral Cortex. Constant source of DHA is Breast Milk.
z Lipotropic Factors are the factors which are required for the synthesis of
Phospholipids. E.g. Essential FAs, Choline, Methionine. Their deficiency leads to
Fatty Liver.
z Cholecystokinin is a Peptide Hormone which is released from Duodenum and
Jejunum in response to Lipid and Protein diet. This hormone helps Gall Bladder to
contract and also cause Pancreas to release it secretions.
z Humans cannot introduce double bonds beyong Δ9 (delta 9) position.
z Antioxidants are added in oils to prevent Rancidity (Hydrolytic cleavage of double
bond leading to formation of Short Chain Fatty Acids & Aldehydes).
LIPOPROTEINS
z Lipids present in Lipoproteins are TGs, Phospholipids, Free Cholesterol
(unesterified) and Cholesterol Ester. TGs and Cholesterol Ester are neutral or totally
Non-Polar and lies in the core. Cholesterol and PLs are Amphipathic Lipids and lie
towards the periphery.
z Proteins present in Lipoproteins are known as Apo Lipoprotein s or Apoproteins
and are synthesized in RER and Golgi Apparatus. They are Apo A-I, A-II, Apo B-48,
Apo B-100, Apo C-I, C-II, C-III and Apo E.
z Lipoprotein s are Chylomicrons, Chylomicron remnant, VLDL (Very Low Density
Lipoproteins), VLDL remnant or IDL (Intermediate Density Lipoprotein s), LDL
(Low density Lipoproteins) and HDL (High density Lipoproteins) in the order of
increasing Density.
z Density is directly proportional to Percentage of Proteins and inversely proportional
to TG Content and Size.
z HDL has maximum density, so smallest size, has minimum TG content and
maximum Proteins.
z In circulation, HDL donates Apo C and Apo E to Chylomicron and VLDL so that
they are converted to Chylomicron remnant and VLDL remnant.
z Chylomicrons are largest in size.
z Lipoprotein Lipase enzyme is synthesized by Adipose Tissues, Cardiac and Skeletal
Muscles. This enzyme is activated by Insulin.
z Chylomicrons carry Exogenous Lipid (or Exogenous TGs) from Intestine to
Peripheral Tissues.
z Chylomicron remnant is degraded in Liver.
Lipids : 57
z Endogenous fats are synthesized by Liver and transported in the form of VLDL. So,
VLDL transports endogenous TGs or Endogenous Lipids from Liver to Peripheral
Tissues.
z Cholesterol is transported to Peripheral Tissues by LDL.
z HDL takes Cholesterol from Periphery to Liver: also known as Reverse Cholesterol
Transport.
z HDL converts Cholesterol to Cholesterol Ester with the help of enzyme LCAT
(Lecithin Cholesterol Acyl Transferase). Apo A-I activates LCAT. LCAT transfers
FA from Lecithin (PL) to cholesterol. Lecithin after losing Fatty Acid becomes
Lysolecithin.
z Main Ligand for LDL-Receptor is Apo B-100 and minor Ligand is Apo E.
z Ligand for HDL-Receptor is Apo A-I.
z Ligand for Chylomicron remnants & VLDL remnants is Apo E.
z Apo C-I and Apo C-II → activate Lipoprotein Lipase.
z Apo C-III → inhibits Lipoprotein Lipase.
z Apo A-I → activate LCAT.
z Apo A-II → inhibits LCAT.
z HDL (cardioprotective) is known as α-Lipoprotein or Lipoprotein -A.
z Deficiency of HDL leads to Tangier’s Disease.
z LDL is known as β-Lipoprotein and is increased in Diabetes Mellitus.
z VLDL is known as Pre- β-Lipoprotein.
z IDL is known as Broad β-Lipoprotein.
z Lipoprotein ‘a’ has a structure similar to Plasminogen, so it interferes with
activation of Plasminogen to Plasmin, leading to Intravascular Thrombosis.
Therefore, it is associated with increased risk for Cardiovascular diseases. It contains
LDL and Apo ‘a’.
z Lipoprotein A or HDL prevent Atherosclerosis.
z Lipoprotein ‘a’ causes Atherosclerosis (as it contains LDL).
z Insulin enhances the action of Lipoprotein Lipase but only in the capillary beds of
Adipose tissue.
z Hormone Sensitive lipase is activated by Glucocorticoids, Glucagon, Epinephrine,
Nor-Epinephrine, Dopamine and Growth Hormones.
z HDL has maximum Electrophoretic Mobility.
z Chylomicron has Apo B-48.
Lipoprotein Lipid Protein

Chylomicron TG Apo B-48
Chylomicron remnant TG + Cholesterol Apo B-48 + Apo E
VLDL TG Apo B-100
VLDL-Remnant/IDL TG + Cholesterol Apo B-100 + Apo E
LDL Cholesterol Apo B-100 + Apo E
HDL Cholesterol Apo-A, Apo-C and Apo-E
58 : Lipids
z Chylomicron remnant has Apo B-48 + Apo E.

z VLDL has Apo B-100.
z VLDL remnant/IDL and LDL has Apo B-100 + Apo E.
z Familial Hyperchylomicronemia or Type I Hyperlipoproteinemia is characterised
by increase in Chylomicrons (↑ ↑) and VLDL (↑ ) and TGs (↑ ↑) while Cholesterol
is normal.
z Familial Hypercholesterolemia or Type II-a Hyperlipoproteinemia is characterised
by increase in LDL (↑ ↑) and Cholesterol (↑) while TGs are normal.
z Familial Hyperlipoproteinemia or Type II-b Hyperlipoproteinemia is characterised
by increase in VLDL (↑) , LDL (↑), Cholesterol (↑) and TGs (↑).
z Dysbeta-Lipoproteinemia or Type III Hyperlipoproteinemia or Broad-Beta
Disease is characterised by increase in Chylomicron remnant (↑ ↑) , V LDL remnant
(↑), Cholesterol (↑ ↑) and TGs (↑ ↑).
z In Type I Hyperlipoproteinemia, there is a defect in Lipoprotein lipase or Apo C-II.
z In Type II-a Hyperlipoproteinemia, there is a defect in LDL receptor or Apo B-100.
z In Type III Hyperlipoproteinemia, there is a defect in Apo E.
z Hypertriglyceridemia is the most predisposing factor for Acute Pancreatitis.
z Tendon Xanthoma means Cholesterol is increased.
z Milky plasma means Chylomicrons are increased.
z Palmar or Tubero Eruptive Xanthoma means Chylomicron remnant and VLDL
remnant are increased.
z Deficiency of LCAT leads to increase in nascent discoidal HDL containing Free
Cholesterol.
z Lipoprotein ‘x’ is an abnormal Lipoprotein found in LCAT deficiency and in
Cholestatic states.
z Complete deficiency of LCAT is known as Norum’s Disease, can progress to end
stage renal disease.
z Partial deficiency of LCAT is known as Fish Eye Disease, which is benign condition.
LIPID METABOLISM
z FA synthesis is an Anabolic pathway and occurs in Fed state and activated by Insulin
hormone.
z Major site of Fatty Acid synthesis is Liver.
z FA synthesis occurs in Cytoplasm.
z Acetyl CoA (produced in Mitochondria) is the starting material for Fatty Acid
Synthesis.
z RLE of Fatty Acid synthesis is Acetyl CoA Carboxylase (adds CO2 to Acetyl CoA to
make Malonyl CoA).
z Malonyl CoA is the activated form in Fatty Acid Synthesis.
z Main enzyme of FA synthesis is Fatty Acid synthase complex/ Palmitate synthase
complex.
z Fatty Acid synthase complex is a Multienzyme complex consisting of 6 enzymatic
activities: Ketoacyl synthase, Malonyl-Acetyl Transacylase, Hydratase, Enoyl
Reductase, Ketoacyl reductase and Thioesterase/Deacylase.
Lipids : 59
e Reducing e
zym nz
y
en m
g Dehydratase
in Malonyl
e
Releasing
ens transacylase Enoyl
enzyme
reductase
nd
Acetyl
Ketoacyl
Co
transacylase
reductase
ACP
Fun
Ketoacyl Thioesterase
divis
ctio
synthase
ion
nal
4`-phospho-
Cys pantetheine
SH Subunit SH
SH division SH
Cys
4`-phospho-
pantetheine
Ketoacyl
synthase
Thioesterase
ACP Acetyl
Malonyl transacylase
Ketoacyl transacylase
reductase Enoyl
Dehydratase
reductase
Fatty acid synthase multienzyme complex
z Fatty Acid Synthase Complex is a dimer consisting of two subunit and each subunit
attached to Acyl Carrier protein (has Sulfhydryl group with Phosphopanetheine).
z Malonyl-Acetyl Tranacylase enzyme is involved in the loading of Acetyl CoA (2C)
and Malonyl CoA (3C).
z Palmitic Acid / Palmitate is the first Fatty Acid synthesized denovo after 7 cycles of
Fatty Acid Synthesis.
z Synthesis of one Palmitate requires 14 NADPH.
z Cofactors required for Fatty Acid Synthesis are NADPH, Biotin & ATP.
z Malate in Cytoplasm gets converted to Pyruvate by Malic enzyme which is a minor
source of NADPH.
Citrate Shuttle
60 : Lipids
z Chain Elongation of Fatty Acids occur mainly in ER and also in Mitochondria.

z Elongation above C10 occurs in ER.
z Desaturation to produce Unsaturated FA occurs in Smooth ER and mainly in Liver.
z Enzyme involved in Desaturation is Membrane Bound Enzyme known as Fatty acyl
CoA Desaturase.
z Most common Desaturase is Δ9-desaturase.
z Humans cannot introduce double bonds beyond Δ9 position.
z Sources of Glycerol-3-Phosphate are DHAP (by enzyme Glycerol-3-P dehydrogenase)
and from Glycerol (enzyme is Glycerol Kinase).
z Glycerol-3-P Dehydrogenase is present in both Liver & Adipose Tissues.
z Glycerol Kinase is present only in Liver.
z Adipose Tissue strictly depends on Glucose Uptake (via GLUT-4) for TG synthesis.
z RBCs always rely on Glucose because they cannot use Ketone Bodies or Fatty Acids
due to Lack of Mitochondria.
z Ketone Bodies are Acetoacetate, β-Hydroxy Butyrate and Acetone.
z Concentration of Ketone Bodies in Blood (normal person) = 0.2 mmol/L
z Ratio of β-Hydroxy Butyrate and Acetone in a Normal person is 1:1 wheras in
Ketosis it become 6:1.
z Ketone Bodies can easily cross IMM.
z Ketone Body Synthesis and Utilization take place in the Mitochondria
z Ketone Body Synthesis occurs in Liver whereas Utilization occurs in Brain, Heart
and Skeletal muscles.
z Ketone Bodies are synthesized when there is increased Lipolysis and increased Acetyl
CoA and decreased Oxaloacetate.
z Ketone Body Synthesis occur during Starvation and Severe Uncontrolled Diabetes
Mellitus.
z Primary Ketone Body /First Ketone Body synthesized– Acetoacetate.
z Secondary Ketone Bodies - β-Hydroxy Butyrate and Acetone.
z Most common Ketone Body found in Blood & Urine is β-OH-butyrate.
z Acetone is volatilized via Lungs and does not act as a Fuel in the body. It is just
released from breath and has diagnostic role.
z Rate Limiting Enzyme of Ketone Body Synthesis is HMG CoA Synthase (enzyme
common to both Ketone Body Synthesis and Cholesterol Synthesis).
z Thiolase is a common enzyme for 4 Pathways : KB synthesis, KB Utilization,
Cholesterol Synthesis, β-oxidation of FA.
z Pathways where HMG CoA is Intermediate: KB synthesis, Cholesterol synthesis and
Leucine Catabolism.
z First enzyme of Ketone Body utilization is Thiophorase/ Succinyl CoA Transferase–
does not use ATP but adds High Energy CoA bond.
z Energy yield from one Acetoacetate is 19 ATPs.
z Energy yield from one molecule of β-OH-butyrate is 21·5 ATPs.
z Rothera’s test is used for detection of KBs and Positive Test indicates Acetoacetate
and Acetone.
z Gerhardt’s test – Detection of KBs – Positive test indicate Acetoacetate (not
Acetone).
z No test available for β-OH-Butyrate.
Lipids : 61
z Liver cannot use KBs because of the absence of Thiophorase.

z RBCs cannot use KB because they lack Mitochondria.
z Neutral Ketone Body – Acetone.
z First enzyme of KB synthesis – Thiolase.
z Immediate precursor of Acetoacetate – HMG CoA.
z Beta Oxidation of FA (odd and even) –in Mitochondria and occurs in Fasting state
or Starvation.
z No ATP produced by Oxidation of VLCFA and α-Oxidation of FA.
z Any defect in β-oxidation leads to Non-Ketotic Hypoglycaemia.
z Ketotic Hypoglycaemia occurs in Von Gierke’s disease.
z Carnitine –Acyl Carnitine Translocase enzyme transports Acyl Carnitine into
Mitochondrial matrix and it also transports Free Carnitine out from Mitochondrial
Matr ix.
z Muscle does not synthesize FAs.
z Malonyl CoA (3C) is the Inhibitor of CPT-I (Carnitine Palmitoyl Transferase-I).
z In Fed state, β-oxidation is inhibited.
z 106 ATPs are obtained by β-oxidation of 16-C Palmitic acid.
z 120 ATPs are obtained by β-oxidation of 18-C Stearic acid.
z 2 ATPs are used in Activation of FA for β-Oxidation.
z Long Chain Acyl CoA Dehydrogenase :starts breaking a Long Chain FA and can
break upto 12C.
z Medium Chain Acyl CoA Dehydrogenase :works after Long Chain Acyl CoA
Dehydrogenase and can do all cleavages below 12C.
z MCAD deficiency is Autosomal Recessive and is the most common Inborn Error of
β-oxidation.
z During Starvation, patient with MCAD deficiency has low Ketone Bodies, and
Hypoglycaemia/Non-Ketotic Hypoglycaemia.
z Ketotic Hypoglycemia occurs in Von gierke’s Disease and in Alcoholism.
z Jamaican Vomiting Sickness occurs after ingestion of unripe fruit of Akee tree which
contains a toxin named Hypoglycin. It inhibits Fatty Acyl CoA Dehydrogenase.
z Generally Fats can never be converted to Carbohydrates but there are two exceptions:
Glycerol and Propionic Acid.
z In Vitamin B12 deficiency, L-Methyl Malonic Acid and Propionic Acid are excreted
in urine.
z In Vitamin B7 deficiency, Propionic Acid is excreted in urine.
z Odd Chain Fatty acids can form Glucose (as they form Propionyl CoA) but Even
Chain Fatty Acids cannot.
z β-oxidation of VLCFA occurs in Peroxisomes upto Octanoyl CoA, Rest in
Mitochondria.
z Zelleweger Syndrome/ Cerebro-Hepato-Renal Syndrome occurs because of the
accumulation of Polyenoic Acids in Brain (containing >C22).
z Zellweger Syndrome is the most severe Peroxisomal Biogenesis Disorder.
z In Zellweger Syndrome, there is accumulation of Phytanic Acid and VLCFA.
62 : Lipids
z Protein Targeting Disorders are I-Cell Disease, Primary Hyperoxaluria, Familial

Hypercholesterolemia, Zellweger Syndrome & Cystic Fibrosis.
z Wolman’s Disease is a Lysosomal Storage Disease but not a Sphingolipidoses.
z Lysosomal Storage Diseases are Mucopolysaccharidosis, Pompe’s Disease,
Sphingolipidoses, Wolman’s Disease, Cystinosis.
z Lysosomal Storage Diseases with Enzyme Replacement Therapy (ERT) are Type
I, Type II & Type VI Mucopolysaccharidosis, Pompe’s Disease, Wolman’s Disease,
Niemann’s Pick Disease, Gaucher’s Disease & Fabry’s Disease.
z X-linked Adreno Leulo Dystrophy/XALD : Defect in the transport of VLCFA
across peroxisomal membrane.
z α-oxidation of FA occurs in Peroxisomes and ER.
z In α-oxidation, there is removal of one carbon from Alpha Carbon atom.
z Refsum’s Disease is defect in Alpha-Oxidation of Fatty Acids and there is
accumulation of Phytanic Acid.
z ω-oxidation of FA occurs in ER.
z In Omega Oxidation, there occurs oxidation of Omega Carbon of Fatty Acid leading
to the formation of Dicarboxylic Acids.
z Cholesterol Synthesis is Anabolic Pathway and occurs in Fed state and is activated
by Insulin.
z Cholesterol is having four fused Hydrocarbon Rings and it is called Cyclo Pentano
Per Hydro Phenanthrene Ring (Steroid Ring).
z Cholesterol has 27 Carbons and one –OH group at Position Number 3.
z All the 27 carbons of Cholesterol are derived from Acetyl CoA.
z Cholesterol is required for Membranes, Steroid Hormones, Lipoproteins, Vitamin
D and Bile Acids.
z RLE of Cholesterol Synthesis – HMG CoA Reductase.
z HMG CoA Reductase catalyzes the conversion of HMG CoA to Mevalonate.
z Cholesterol Synthesis occurs in all tissues. Acetyl CoA is starting material and
NADPH is used for Reductive Biosynthesis. ATP is also used.
z First two steps in Cholesterol Synthesis are same as those in KB synthesis but
Cholesterol Synthesis takes place in Cytoplasm.
z HMG CoA can be converted to Mevalonate, Cholesterol, Ketone Bodies & Acetyl
CoA.
z Compounds derived from Cholesterol are Bile Acids, Vitamin D and Steroid
Hormones.
z Primary Bile Acids are Cholic Acid & Chenodeoxy Cholic Acid.
z Secondary Bile Acids are Deoxycholic Acid & Litho Cholic Acid.
z Rate Limiting Enzyme of Bile Acid Synthesis is 7-α Hydroxylase (requires
Vitamin C).
z Ursodeoxy Cholic Acid is a Secondary Bile Acid, used in the treatment of Liver
diseases.
nnn
CHAPTER
MOLECULAR 7
CHEMISTRY & METABOLISM OF NUCLEOTIDES
z Nucleic Acid is polymer of Nucleotides.
z Nucleotide = Nitrogenous base + Sugar +
Phosphate.
z Nucleoside = Nitrogenous base + Sugar.
z Purines are Adenine and Guanine. Purines Vs Pyrimidines

z Pyrimidines are Cytosine, Uracil and
Thymine.
z Thymine present only in DNA, not in RNA.

z Uracil present only in RNA, not in DNA.
z Thymine is a Pyrimidine Nitrogenous base. Thiamine is a Vitamin (Vitamin B1).
Vitamin B1 has a Pyrimidine like Ring in its structure.
z Guanidine is a Protein Denaturating Agent but Guanosine is a Nucleoside of
Guanine (i.e. Guanine + sugar).
z Most abundant Free Nucleotide in Mammalian cells is ATP.
z Conjugated Double Bonds in the rings is responsible for Absorption of UV light.
z DNA absorbs UV-Light at 260 nm (UV light is Electromagnetic Radiation with a
wavelength from 10 nm to 400 nm, shorter than that of Visible light and longer than
X-rays).
z Amino Acids absorbs UV-Light at 280 nm.
z NAD/NADP absorbs UV-Light at 340 nm.
z Porphyrins absorbs UV-Light at 400 nm.
z Sugar in Nucleic Acids is always a Pentose. This pentose can be Ribose or Deoxy-
Ribose.
z Ribose exists in Furanose form. At 2’ position, -OH group is present.
z If oxygen is removed from 2’ position, then it is known as 2’ Deoxyribose.
z If oxygen is removed from 2’ & 3’ position, then it is known as 2’3’ Di-deoxyribose.
64 : Molecular
z NADPH is the ultimate donor of Reducing Equivalents for the conversion of

Ribonucleoside Diphosphate to its Deoxy Ribonucleoside Diphosphate (Enzyme –
Ribonucleotide Reductase).
z Two pathways for synthesis of Purine Nucleotides are De Novo Pathway and
Salvage Pathway.
z De Novo Pathway is High Energy Consuming Pathway and is most active in Liver.
z Phosphoribosyl Pyrophosphate (PRPP) Synthetase catalyze the Synthesis of PRPP
from Ribose-5-P.
z Commited and Rate Limiting Step of Purine Synthesis in De Novo Pathway is
conversion of PRPP to Phospho Ribosyl Amine (PRA) by PRPP Glutamyl Amido
Transferase.
z First Purine Nucleotide to be synthesized is IMP (Inosine Mono Phosphate).
z Parent Purine Nucleotide for AMP and GMP is IMP (Inosine Mono Phosphate).
z AMP synthesis require Aspartate and GTP.
z GMP synthesis require Glutamine and ATP.
z 6-Mercapto Purine is an Inhibitor of Adenylosuccinate Lyase.
z PRPP is used in Purine Synthesis, Pyrimidine Synthesis, Histidine Synthesis and
Niacin Synthesis.
z Salvage Pathway for Purine Synthesis occurs in RBCs,WBCs, Brain and Bone
Marrow.
z Inhibitors of Purine Synthesis: PABA (Para-Amino Benzoic Acid) Analogues and
Antifolates.
z Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) is enzyme of
Salvage Pathway which converts Hypoxanthine to IMP and Guanine to GMP.
z Complete deficiency of HGPRT leads to Lesch Nyhan Syndrome (Gout & Self
Mutilation).
z Partial deficiency of HGPRT leads to Kelley Seegmiller Syndrome (only Gout).
z Adenine Phospho Ribosyl Transferase (APRTase) converts Adenine to AMP.
z Adenosine is the only Purine Nucleoside which can be salvaged i.e. Adenosine gets
converted to AMP by Adenosine kinase.
z Adenosine Deaminase (ADA) converts Adenosine or Adenosine-5-Phosphate to
Inosine or Inosine-5’-P. This enzyme is very important in B and T- lymphocytes and
NK cells.
z Increased ADA levels is highly suggestive of Tuberculosis.
z Deficiency of ADA leads to SCID (Severe Combined Immuno Deficiency)-
Adenosine (substrate) is increased which gets converted to Deoxy ATP, which is
inhibitor of Ribonucleotide Reductase. So, synthesis of Deoxyribonucleotides stops
& Cell cannot divide due to lack of DNA Synthesis.
z SCID was the first disorder to be treated by Gene Therapy.
z Xanthine Oxidase: Rate Limiting Enzyme of Purine Catabolism.
z Xanthine Oxidase (RLE) catalyzes the conversion of Xanthine to Uric Acid and
requires Molybdenum.
z Allopurinol is the Suicide Inhibitor of Xanthine Oxidase.
Molecular : 65
z Catabolic End Product of Purines in Primates, Amphibians, Reptiles and Birds is

Uric Acid.
z In Non-Primate Mammals, Uricase /Urate Oxidase forms Allantoin as the End
Product of Purines.
z Combined Mechanism of Hyperuricemia (increased synthesis of Uric Acid and
decreased excretion): Von Gierke’s Disease, Aldolase B Deficiency, Shock and
Alcohol.
z HGPRT Deficiency → Increased Uric Acid.
z Xanthine Oxidase Deficiency → Decreased Uric Acid.
z PRPP Synthetase Deficiency → Decreased Uric Acid.
z Low Purine Diet is Milk (specially cow’s milk), Yoghurt, Cheese and Vit C - help in
excretion of Uric Acid from Kidneys.
z High Purine Diet is Spinach, Mushrooms, Peas, Cauliflower, Meat, Fish (tuna),
Liver.
z Gout Patients should avoid Heavy exercise & Alcohol as it leads to Lactic Acidosis
& Hyperuricemia.
z First Pyrimidine Nucleotide to be synthesized is OMP (Orotidine Mono Phosphate).
z Rate Limiting Step of Pyrimidine Synthesis in Eukaryotes is CPS-II (Carbomoyl
Phosphate Synthetase-II).
z Rate Limiting Step of Pyrimidine Synthesis in Prokaryotes is Aspartate Trans
Carbamoylase.
z OPRT and Decarboxylase are Bifunctional Enzymes (two enzyme activities on a
single protein). This enzyme/protein is known as UMP Synthase.
z UTP gets converted to CTP by enzyme CTP Synthetase (requires energy from ATP)
and requires Amino group from Glutamine.
z Orotic Aciduria is a rare Autosomal Recessive Disorder, which is defect in UMP
Synthase.
z Orotic Aciduria is the most common Metabolic Error in Pyridimine Synthesis.
z In Type I Orotic Aciduria, both OPRT and Decarboxylase enzymes are deficient.
z In Type II Orotic Aciduria, only Decarboxylase is deficient.
z Megaloblastic Anaemia occurs in Orotic Aciduria due to deficiency of Pyrimidines.
z UDP (Uridine Diphosphate) Sugars are used in synthesis of Glycogen, Galactose,
Glycoprotein etc. & serve as carrier of Activated Intermediates e.g. UDP-Glucose,
CDP-Choline.
z UDP-Glucuronic Acid is used for the Conjugation Reactions (like Bilirubin),
Synthesis of Proteoglycans.
z PAPS (Phospho Adenosyl Phospho Sulphate) act as Sulphate Donor.
z Cyclic AMP and Cyclic GMP act as Second Messengers.
z NAD, NADP, FAD, FMN act as Coenzymes.
z DNA and RNA are Nucleic Acids (Polymer of Nucleotides).
z Hershey & Chase Experiment was done on Bacteriophages and it proved that DNA
is the Genetic Material in organisms.
z DNA is Double Stranded in both Eukaryotes and Prokaryotes.
z In DNA, Purines always equals Pyrimidines.
z RNA is Single Stranded, so Purines are not necessarily equal to Pyrimidines.
66 : Molecular
z DNA is Helical (right handed) and the two strands are Antiparallel.
z DNA is made up of four Nitrogenous bases : Adenine (A), Thymine (T), Guanine
(G) and Cytosine (C).
z B-DNA and A-DNA are Right Handed whereas Z-DNA is Left Handed.
z B-DNA is the major DNA in cells.
z Z-DNA has a function in regulation of Gene Expression, particularly in GC sequence
(alternating Purine-Pyrimidine).
z Gene Expresssion = Transcription + Translation.
z Thymine (T) is present instead of Uracil (U) in DNA to prevent Mutations.
z Cytosine gets converted to Uracil (by removal of amino group) very spontaneously
in DNA, by enzyme Cytosine Deaminase.
z Main difference between DNA and RNA is Sugar i.e always Deoxyribose in DNA
and its always Ribose in case of RNA.
z Chargaff’s Rule: Adenine must pair with
Thymine and Guanine with Cytosine with
weak Hydrogen bonds. The number of
Purines (A+G) is equal to the total number
of Pyrimidines (C+T) but the number
of (A+T) is not equal to (G+C), it can be
variable.
z Nucleosomes = DNA + Proteins.
z DNA is negatively charged due to Phosphates.

z Prokaryotes have circular double stranded DNA whereas Eukaryotes have linear
double stranded DNA.
z Histone Octamer contains H2A, H2B, H3 and H4 Histone proteins.
z Linker / Spacer DNA is present in between two Histone Octamers. It is associated
with Linker Histones i.e H1 and H5.
z Histone Proteins have positive charges because they are rich in Basic Amino Acids
(Lysine, Arginine).
z Due to opposite charge on DNA and Histone Proteins, they get tightly bound
forming Nucleosomes.
z If DNA is separated from Histone, then it is free or is transcriptionally active.
z PTMs (Post Translational Modifications) of Histones: These are reversible covalent
modifications in which N-terminal of Proteins is modified by adding or removing a
group and helps in regulation of Gene Expression.
z Various ways of Histone Modifications are: Acetylation, Phosphorylation,
Methylation, ADP Ribosylation, Mono-Ubiquitylation and Sumoylation.
Molecular : 67
z Acetylation and Phosphorylation of Histones lead to increased Euchromatin

Formation → Gene Activation.
z Deacetylation and Dephosphorylation of Histones leads to Gene Inactivation.
z Histone Methylation leads to Gene Inactivation.
z DNA present in compact form along with Histones is known as Chromatin and
exists only in Eukaryotes.
z During Mitosis, Chromatin is heavily condensed but during Interphase, it is less
condensed.
z Euchromatin is loose DNA and genes are active.
z Heterochromatin is tightly bound DNA and genes are inactive.
z The interconversion of Euchromatin and Heterochromatin is known as Chromatin
Remodelling.
z Proteins and DNA are bound by Weak bonds such as Hydrogen, Ionic, Vanderwaals
and never by Covalent bonds.
z The bonds present in DNA are Hydrogen bonds, 3’ 5’ Phosphodiester bonds and
β-N-Glycosidic bonds.
z 3’ 5’ Phosphodiester bonds are formed between 3’OH of previous Nucleotide with
5’ Phosphate of incoming Nucleotide.
z At 5’end of a Nucleic Acid, there is always a Free Phosphate present.
z At 3’end of a Nucleic Acid, a Free –OH is always present.
z New Nucleotide is always added at the 3’end of a Nucleic Acid.
z Synthesis always occurs in 5’ to 3’ direction.
z Reading & Writing of Base Sequence is always done in 5’ to 3’ direction.
z Nucleases are enzymes which break the Phosphodiester bond present between the
Sugar and Phosphate in a Nucleic Acid.
z Nucleases are of two types: Exonucleases and Endonucleases (or Exci-Nuclease).
z Exonuclease cut the Nucleic Acid from the sides either from 5’ end or from 3’ end.
z 5’ → 3’ Exonuclease starts cutting from 5’end and moves from 5’ to 3’ direction.
z 5’ → 3’ Exonuclease starts cutting from 3’end and moves from 3’ to 5’ direction.
z Restriction Endonucleases (molecular scissors) cut the Nucleic Acid at specific
site (the pallindromes) known as Pallindromic Sites. The fragments formed
after digestion by Restriction Endonuclease Enzyme, are known as Restriction
Fragments.
z Pallindromes are same sequences present on opposite strands of DNA, read in 5’
→ 3’ direction.
z DNA Methylation at CG sites is the most common method of Gene Inhibition in
Genomic Imprinting.
z In mammals, 70-80% of CpG Cytosines are Methylated.
z CpG Islands usually has G+C content greater than 50%.
z Majority of Gene Promoters have high CG content.
z In Non-Hematopoetic cells, DNA Methylation is done to inhibit Transcription of
Globin chain genes.
z In Hematopoetic cells, genes are Hypomethylated.
z The Diploid Human Genome consists of 23 pairs of Chromosomes (composed of
20-25 thousand genes).
68 : Molecular
z Haploid set of Chromosomes is estimated to be 3·2 × 109 Base Pairs.

z Genes consisting of DNA base pairs are located on Chromosomes.
z A Gene is a sequence of Base Pairs that produces a functional product including
RNA molecule and subsequently a Peptide.
z The Diploid Genome contains two alleles of each gene.
z An Allele is positioned on a locus (specific location of a gene or DNA sequence on
a chromosome).
z Chromosomes 1 to 22 are called Autosomes and the 23rd pair is Sex Chromosomes
i.e. X and Y.
z Homologous Chromosomes
are Chromosome Pairs (one
from each parent) that are
similar in Length, Gene Position,
and Centromere location.
The position of genes on each
Homologous Chromosome is
exactly same, however, the genes
may contain different alleles.
z Sister Chromatids are exact copy of each other.

z Centromere is a constricted
point of the chromosome.
z Telomeres are ends of
chromosomes.
z Exons are the genes which give
rise to proteins.
z Introns are Intervening
Sequences which are present in
between Exons.
z Single Nucleotide Polymorphisms (SNPs) is the most common type of

Polymorphism in which Single Nucleotide is variable in different population.
z Repeat Length Polymorphism: Tandem repeats of a particular length DNA occurs
and the number of these repeats are variable in different population.
z Repetitive Sequence in DNA (Satellite DNA) are often clustered in Centromeres &
Telomeres.
z Short Tendem Repeats/STRs are known as Microsatellites and the repeat size is
2-6 base pairs.
z Micro-satellites or STRs are widely used for DNA Profiling in Cancer Diagnosis
(specially Colorectal Cancer). In cancer cells, Microsatellites are gained or lost at
Molecular : 69
high frequency during each round of Mitosis. So Tumor Cell gives a different Genetic
Fingerprint than the host cell.
z Variable number of Tandem repeats/VNTRs are also known as Mini-Satellites and
the repeat size is 15-70 base pairs.
z Mitochondrial DNA: same like Prokaryotic
DNA i.e. Circular double stranded and no
Introns present.
z Mitochondrial (DNA/mtDNA) is
synthesized by γ-Polymerase (gamma).
z mtDNA has high rate of Mutation as
compared to Nuclear DNA.
z mtDNA contains 16500 base pairs and 67
genes, all of which are essential for normal
Mitochondrial function.
z Thirteen of these 67 genes provide
instructions for making enzymes involved
in oxidative phosphorylation.
z AUA Codon codes for Isoleucine but in mitochondrial DNA, it codes for
Methionine.
z UGA Codon is a stop codon but in mitochondrial DNA, it codes for Tryptophan.
z AGA & AGG codes for Arginine but in mitochondrial DNA, these are Stop Codons.
z Mitochondrial inheritance is Non-Mendelian & it is only transmitted through
Mother.
z Mutations in mt DNA mostly affect Oxidative Phosphorylation and there is
decreased ATP in cells.
z Diseases related to Mutations in mtDNA: MELAS, Kearns syndrome, Leber
Hereditary Optic Neuropathy, Leigh syndrome & NARP syndrome.
z MELAS : Mitochondrial Encephalopathy, Lactic Acidosis and Stroke like Episodes.
z Kearns/Sayre Syndrome : Large deletion of mt DNA. Patient has Ophthalmoplegia
and Retinopathy.
z Leber Hereditary Optic Neuropathy : Optic Nerve affected leading to Vision Loss.
z Leigh Syndrome : Brain affected leading to Developmental Delay, Muscle Weakness.
z NARP Syndrome : Neuropathy, Ataxia & Retinitis Pigmentosa.
z DNA Recombination or
Recombination DNA
Molecular Cloning is the
insertion of DNA fragments
to produce new Nucleotide
+
sequence arrangements.
70 : Molecular
z Types of DNA Recombination:

Homologous Recombination (HR)
Site Specific Recombination (SSR) or Non-homologous recombination
Transposition
z Site Specific Recombinase are the enzymes that catalyze DNA Exchange (direction
sensitive) between Short Target Site sequences (30- 40 nucleotides.) that are specific
to each Recombinase.
z Examples of Site Specific Recombinase are CRE Recombinase, λ Phage encoded INT
protein , Yeast FLP Recombinase
z CRE/Lox Recombinase associates specifically with the Lox P locus.
z Transposition is highly specialized form of recombination in which a segment of
DNA moves from one location to another, either on the same chromosome or a
different chromosome.
z Transposable Elements are known as Jumping Genes as these are DNA sequences
that can move from one chromosome locus to another.
z There are two types of Transposable Elements:
Transposons
Retrotransposons
z Transposons move by means of DNA Intermediate.
z Retro Transposons move by means of an RNA Intermediate.
z Small Scale Mutations (Micro Alteration) or Gene Mutation are Point Mutations,
Insertions and Deletions.
z Point Mutations involve Transition and Transversion.
Transitions - Pyrimidine to Pyrimidine or Purine to Purine
Transversions - Pyrimidine to Purine or Purine to Pyrimidine
z Each Amino acid has more than one codons, is known as Degeneracy or Redundancy.
z Point mutations are of three types namely Silent, Missense and Non Sense Mutations.
z In Silent Mutation, Primary Structure of Protein always remain the same. Codon is
replaced by another codon which codes for same Amino Acid.
z Silent Mutations occur due to Degeneracy of Codons (codon is changed but Amino
Acid is not changed). So Degeneracy prevents Mutations.
z In Non Sense Mutation, Primary Structure of Protein will be changed, as there will
be premature Termination of Protein Synthesis due to introduction of Stop Codon.
z In Missense Mutation, a Codon is replaced by another Codon coding for a different
Amino Acid.
z Trinucleotide Repeat Expansion: a sequence of three bases that is repeated in
tandem will become amplified in number, so that too many copies of the triplet
occur.
z Diseases due to Trinucleotide Repeat Expansion:
Hungington Disease
Fragile X Syndrome
Frame Shift Mutation
Splice Site Mutation

Molecular : 71
z Huntington Disease: Neurogenerative - Trinucleotide Repeat Expansion of Codon

for Glutamine, leads to formation of an abnormal Protein which is degraded easily
producing Toxic Fragments that aggregate in Neurons.
z Fragile X Syndrome: Trinucleotide Repeat Expansion leading to Hypermethylation
which cause Gene Silencing.
z Frame Shift Mutation: Addition or Deletion of Nucleotides (not divisible by 3) alter
the reading frame.
z Splice Site Mutation: Can interfere in the removal of Introns from the Primary
Transcript.
z Large Scale Mutations (Macro alteration) or Chromosomal Mutations include
Amplification/Duplication, Deletions, Inversions, Translocations and Aneuploidy.
z An Inversion that involves the Chromosome’s constriction point (Centromere) is
called a Pericentric Inversion.
z An Inversion which occurs in the Long arm (q) or the Short arm (p) and does not
involve the centromere is called a Paracentric Inversion.
z Translocation occurs when a piece of one chromosome breaks off and attaches to
another chromosome. It can be Balanced or Unbalanced.
z If no genetic material is lost or gained from the Chromosome, then called Balanced
Translocation. This occurs between the Homologous Chromosomes.
z If there is a gain or loss of genetic material from the chromosome. Then called
Unbalanced Translocation.
z In Robertsonian Translocation, there is joining of long arms of two chromosomes.
The tiny short arms are usually lost.
z Aneuploidy is abnormal Chromosome Number. It can be Trisomy (three
chromosomes instead of two) or Monosomy (one chromosome).
DNA REPLICATION
z Central Dogma of Molecular Biology: The flow of information from DNA to RNA
and then to Proteins.
z Reverse Transcription is the synthesis of DNA from RNA by enzyme RNA
Dependent DNA Polymerase.
z Reverse Transcription occurs in Retroviruses such as HIV virus, Transposons and
Telomerase.
z DNA Replication and Synthesis of Histones occurs in S-phase of Cell Cycle.
z DNA Replication is Semi-Conservative in nature (one parental strand is conserved
each time).
z Prokaryotes have one Origin of Replication (ori) while Eukaryotes have multiple
ori sites.
z Ori is AT rich sequence and can be separated easily.
z DNA Replication is bidirectional (Replication moves in both directions from the
ori).
z The Temperature at which half of the Helical Structure is lost is known as Melting
Point (Tm).
72 : Molecular
z High GC content of DNA raises Tm.

z Enzymes of DNA Replication: Helicases, Topo-isomerases, SSBs (single strand
DNA Binding Proteins), Primase, DNA Polymerase III, DNA Polymerase I, and
DNA Ligase.
z Helicases cause strand separation using ATP and creates Positive Supercoils.
z Overwinding is known as Positive Supercoiling.
z Underwinding is known as Negative Supercoiling.
z Topo-Isomerases cut and reseal DNA and relieves Positive Supercoiling without the
use of ATP and gain energy from the cleavage of Phosphodiester Bonds.
Type I → Topo-Isomerases cut one strand.
Type II → Topo-Isomerases cut two strands.
DNA Gyrase is a counterpart of Topo-Isomerase-II in Prokaryotes. This enzyme
uses ATP and introduces Negative Supercoils.

z Helicases and Topo-Isomerases work in tandem to do strand separation.
z Single Stranded DNA Binding proteins (SSBs) prevent Reannealing or rejoining
of two separated strands as they have high affinity for single strand and also protect
from Nucleases attack. These are not Enzymes.
z RPA (Replication Protein A) → In Eukaryotes, for prevention of Reannealing.
z Helicase, Topo-Isomerases and SSBs → Together known as Unwinding Proteins.
z Primase synthesize Primers (RNA) from Template DNA.
z DNA Dependent RNA Polymerase in Replication is Primase.
z In Eukaryotes, α-Polymerase synthesize primers.
z In Prokaryotes, DNA-G protein synthesize primers.
z The Template or the Parent strand is
3'
read from 3’ to 5’ direction. 5'
z The strand synthesized in one stretch
Leading
is known as Leading strand. strand
z The strand synthesized in short 3' 5'
fragments (Okazaki fragments) is 3
5'
known as Lagging strand. Lagging
strand
z Only one Primer is needed for 3' Okazaki
Leading strand but multiple Primers 5' fragments
are needed for Lagging Strand.
z DNA Polymerase III synthesize both Leading and Lagging strands. Substrate for
this enzyme is Deoxyribo Nucleotide Triphosphates (dNTPs).
z DNA Polymerase III has Polymerase (5’→3’ Polymerase) Activity & 3’→5’
Exonuclease (Proofreading Activity).
z DNA Polymerase III synthesizes Okazaki Fragments.
z DNA Polymerase II has 3’→5’ Exonuclease (Proofreading Activity) & Repair.
z DNA Polymerase I fills the gap between Okazaki Fragments.
z If 2’3’ Dideoxy Ribose is used, DNA synthesis stops.
Molecular : 73
z If any of the four substrates i.e dATP, dGTP, dCTP, dTTP is not available, DNA
synthesis stops.
z DNA Polymerase I remove RNA Primers from both Leading and Lagging strand.
z DNA Polymerase I has Polymerase (5’→3’ Polymerase) activity and 3’→5’
Exonuclease (Proofreading Activity) & 5’→3’ Exonuclease Activity.
z Klenow Fragment is a fragment of DNA Polymerase I which has 5 → 3’ Polymerase
Activity and 3’ → 5’ Exonuclease Activity.
z DNA Ligase acts only on the Lagging strand and creates 3’5’ Phosphodiester bond
using ATP.
z Telomeres have tandem repeats of a transcriptionally inactive Hexameric sequence
(TTAGGG)n.
z Telomeres provide structural support to the Chromosome, prevent attack by
Nucleases and distinguish a true end of Chromosome from break in dsDNA.
z Telomeres shorten as cell divides.
z Telomerase enzyme provides longevity to cells.
z Telomerase is Ribonucleoprotein i.e. it has RNA + Protein.
z Telomerase is not a Ribozyme because this RNA is not used as enzyme. It is used as
a template for DNA synthesis.
z Telomerase is RNA Dependent DNA Polymerase or Reverse Transcriptase.
z Telomerase activity increases in Cancer and decreases in Aging.
z Germ cells have more Telomerase Activity than Stem Cells.
z Proofreading is correction during Synthesis, occurs in S-phase.
z DNA Repair is correction after Synthesis.
z Proofreading has 3’→5’ Exonuclease Activity.
z Proofreading and DNA repair both are done by DNA Polymerase II in Prokaryotes.
z In Eukaryotes, all Enzymes can do Proofreading except α and β-Polymerase.
z Most Repairs occur in G1 phase of Cell Cycle. But Mismatch Repair occurs in G2
phase.
z DNA Repair is done mainly by β-polymerase in Eukaryotes.
z ε-polymerase has a minor role in DNA repair. Also has role in Leading Strand
synthesis in case of Eukaryotes.
z δ-Polymerase in Eukaryotes – Lagging Strand Synthesis.
74 : Molecular
Comparison of prokaryotic and eukaryotic

DNA polymerase
E. coli Eukaryotic Function
I z Remove primer and fill the gap
II z Remove primer and fill the gap
β z DNA repair
γ z Microchondrial DNA synthasis
III ε z Leading strand synthesis

δ z Lagging strand synthesis
DNA-G α z Primase
z Primers are removed by RNase, FEN-1 (Flap Endonuclease) and δ-Polymerase

(minor role).
z Norfloxacin, Ciprofloxacin inhibit DNA Gyrase.
z Arylhydrazinopyrimidines inhibit DNA Polymerase III.
z Ethylmaleimide, Aphidicolin, Butyl-Phenyl-dGTP inhibit Enzymes of DNA
Replication.
z Didanosine (2’3’dideoxyinosine), Cytarabine, Vidarabine, Zidovudine, Acridine,
Ethidium, Actinomycin, Mitomycin are the inhibitors that directly interact with
DNA.
TRANSCRIPTION & TYPES OF RNAs

z There are three types of RNAs :
mRNA
rRNA
tRNA
z mRNA is the most Heterogenous RNA, synthesized in Nucleolus.

z rRNA (ribosomal) is the most abundant and is synthesized in Nucleolus.
z tRNA (transfer) is the smallest and has maximum Modified Bases e.g. Pseudouracil.
z Both mRNA and tRNA are synthesized in the Nucleus.
Molecular : 75
z tRNA and rRNA have uncapped 5’

ends and no Poly A tail at 3’ends.
z tRNA and rRNA are not translated.
z tRNA is the Adaptor to carry
specific Amino Acids.
z tRNA has T-arm/TΨC arm/
Pseudouridine arm which helps
in non covalent binding of A-site
of ribosome during initiation and
elongation.
z DHU arm/D arm/Dihydrouridine
arm selects the correct amino acid
by recognizing specific Amino Acyl
tRNA Synthetase enzyme.
z Codon is present on mRNA
(messenger) and Anticodon is
present on tRNA.
z Amino acid is covalently attached at the 3’end of tRNA.
z mRNA which is formed from more than one gene is known as Polycistronic and is
a characteristic of Prokaryotes.
z In Eukaryotes, each mRNA comes from a single gene knwn as Monocistronic
mRNA.
z Bacterial/ Prokaryotic Ribosome: 70S comprising larger subunit 50S (5s, 23s
rRNA) and smaller subunit 30S (16s rRNA).

z Eukaryotic Ribosome: 80S comprising larger subunit 60S (5s ,5.8s, 28s rRNA) and
smaller subunit 40S (18s rRNA).

z Exons or Coding DNA sequence are only 1-2% of all Genome. Rest is Non-Coding.
z Presence of Introns prevent Mutations.
z Introns are transcribed and not translated.
z Only eukaryotic DNA have introns.
z Coding RNA is only mRNA. Rest are Non Coding RNAs.
z The final product of Gene Expression can be a Protein or RNA, depending on the
Gene.
z Ribozyme means RNA act as Enzyme.
z Substrate of Ribozyme is mostly RNA.
z Ribozyme catalyzes the breakage and synthesis of Phosphodiester bond and no ATP
is used.
z Telomerase and RNase H are not Ribozymes.
z Ribozymes : rRNA, Splicing Ribozyme (snRNA), Ribonuclease P.
z Ribonuclease P cleaves tRNA.
z Transcription is the synthesis of RNA from DNA by enzyme RNA Polymerase
(DNA Dependent RNA Polymerase).

76 : Molecular
z Transcription has higher error rate than Replication.
z RNA Polymerase does not require RNA polymerase

Primer and cannot do Proofreading.
z RNA Polymerase activity in 5’→3’
direction.
z Holoenzyme of RNA Polymerase
contains 5 subunits- σ ααββ’ω.
z α, ω subunit is required for Enzyme
Assembly.
z β subunit has Catalytic Activity.
z β’ subunit is required for Template binding and σ for Initiation/Recognizing

Promoter.
z ααββ’ω make the Core Enzyme represented as ‘E’.
z E + σ subunit makes the Holoenzyme.
z Prokaryotes have single RNA Polymerase.
z Types of RNA Polymerase in Eukaryotes:
Type I – synthesize all rRNA except 5s rRNA
Type II – synthesize mRNA, mi-RNA, inc-RNA, few snRNA, snoRNA
Type III – synthesize 5s rRNA, tRNA, few snRNA and snoRNA
Mitochondrial RNA Polymerase – synthesize Mitochondrial RNA
z Template Strand → Strand where gene is present, also known as Anti-Sense/ Non-
Coding/ Minus strand.
z Non Template Strand → also known as Sense/ Coding/ Plus strand.
z RNA Polymerase first binds with the Promoter Region (helps in initiation but
promoter region is not transcribed).
z TATA box in Prokaryotes known as Pribnow Box present at -10 position on DNA
(10 base pairs upstream to the transcription start site).
Molecular : 77
z TATA box in Eukaryotes known as Hogness box present at -25 position on DNA
(25 base pairs upstream to the Transcription Start Site).
z -35 sequence → present only in Prokaryotes, 35 base pairs Upstream to the

Transcription Start Site.
z CAAT box /-75 sequence → in Eukaryotes, present 75 base pairs Upstream to
Transcription Start Site.
z Regulatory Sequences are present on coding strand in 5’ →3’ sequence.
z The first base at Transcription Start Site is given +1 number. There is no base
designated as ‘0’.
z The bases to the left or towards 5’end are assigned negative number or called
Upstream.
z Substrate for Elongation step in Transcription is Ribonucleotide Triphosphates.
z Two high energy Phosphates used to add one Ribonucleotide.
z Termination of transcription may be Rho-dependent (ρ) or Rho-independent.
z Rho-dependent Termination requires ρ-protein and uses ATP and Helicase activity.
It releases the RNA at Termination Site.
z Inhibitors of Transcription are Actinomycin D, Rifamycin and α-Amanitin.
z Enhancers of Transcription increase the rate of initiation and can be located
upstream or downstream of promoter.
z Three Post Translational Modifications occur in all eukaryotic mRNA. These are
also known as RNA processing. These are Cap at 5’end, removal of Introns/ Splicing
and Tail at 3’end.
z 7-Methyl Guanosine Cap is added at 5’end of mRNA by Guanylyl transferase by
unusual 5’5’ Triphosphate Linkage.
z Methyl group is donated by SAM by enzyme Guanine 7 methyl transferase (occurs
in cytoplasm).
z Alternate Splicing and RNA Editing are exceptions to One Gene One Protein
theory.
z Cap facilitates the initiation of Translation and stabilizes mRNA by preventing it
from attack by 5’ Exonucleases.
78 : Molecular
z At 3’end, a Poly A Tail is added by enzyme Polyadenylate Polymerase.

z Splicing Ribozyme → Self Splicing Introns and Spliceosomal Introns.
z Immature mRNA has both Intron &
Exon.
z Mature mRNA contains only Exons
z Lariat Formation occurs in Group II
Self Splicing Introns and Spliceosomal
Introns.
z snRNA (small nuclear) which takes part in Splicing → U1,U2,U4,U5,U6.
z snRNA have unique 5’ caps. Sm-class snRNA have 5’ Trimethyl Guanosine Caps, &
Lsm-class snRNA have 5’ Monomethyl Phosphate caps.
z Differential RNA Processing/ RNA Editing is a mechanism to produce diverse set
of Proteins from a limited set of Genes.
z Transcription and Post Transcriptional Modifications occur in Nucleus but in case
of rRNA, they occur in the Nucleolus.
z Regulatory Sequences (RS) are present in Non-Coding Region of DNA and interact
with Regulatory Molecules (RM) or Transcription Factors to activate or repress
Genes.
z RS are cis-acting if they control Gene Expression of a Gene that lies on the same
chromosome as the RS.
z RS are trans-acting if they are produced from a particular Gene from a Chromosome
but they affect the Regulation of a Gene which is lying on the other Chromosome.
z Protein Motif is that modified portion of a Regulatory Protein which binds to
Response Element on DNA.
z Response Element is a DNA sequence which gives a site of Protein Binding. At this
site of DNA, Protein Motif binds.
z Helix Turn Helix Protein Motif: Regulate genes of development.
z Zinc Finger Protein Motif : e.g. Steroid Hormone Receptors.
z Helix Loop Helix Protein Motif: Regulate Immune System Genes.
z Leucine Zipper: Regulate Cell Division.
OPERON MODEL
z Operator is a site on DNA that regulate the activity of the Gene by binding a Protein
known as Repressor.
z Inducer can bind to Repressor and can remove Repressor from Operator site leading
to Gene Activation. Inducer is Lactose in Lac Operon.
z Operon means a complete unit operating on itself. It has the capability to activate or
inhibit the Genes.
z Operons are found in Prokaryotes.
z O-site : Operator lies downstream to Promoter.
z CAP site : lies Upstream of Promoter. CAP-cAMP Complex (trans acting protein)
binds here.
z Lac Operon Genes are activated if glucose decreases in the environment of E.Coli
and if Lactose is present.
Molecular : 79
z Lac I (Inhibitory) is a House Keeping Gene i.e. always active and has its own
Promoter.
z Lac I produces Repressor Tetramer (a trans acting factor).
z Lac Z codes for β-Galactosidase – breaks Lactose to Galactose and Glucose.
z Lac Y codes for Permease – Facilitate permeation of Lactose into the cell.
z Lac X codes for Transacetylase - Acetylates Lactose.
TRANSLATION
z Codons are Nucleotide Triplets (3 nucleotides make 1 codon).
z 4 Nucleotide Bases used to produce three base Codons i.e. A, U, C, G.
z Total number of Codons possible = 64.
z STOP Codons : UAA, UAG, UGA – do not code for any Amino Acid.
z Methionine and Tryptophan have only 1 Codon i.e. do not show degeneracy.
z Genetic Code is Unambiguous : each Codon is specific for its Amino Acid e.g. AUG
will always Code for Methionine.
z Genetic Code is Universal i.e well conserved during evolution with only slight
differences.
z Genetic Code is Non-Overlapping and Commaless.
z There is Antiparallel binding between Codon and Anti-Codon in tRNA.
z Wobble Hypothesis: Movement of first base of Anti-Codon can occur to allow non-
traditional base pairing with the last base of Codon. This movement is called Wobble,
which allows single tRNA to recognize more than one Codon.
z Ribosomes are spherical bodies made up of RNA and Proteins. They are not
considered Organelles as they are Non-Membranous.
z Mitochondria contain their own Ribosome (55s) and their own unique circular
dsDNA.
z mRNA is translated in 5’→3’ direction and Proteins are synthesized from Amino
terminal to Carboxy terminal.
z Activation of Amino Acids or Charging of tRNA to make Aminoacyl tRNA is done
by enzyme Amino Acyl tRNA Synthetase.
z The enzyme Amino Acyl tRNA Synthetase is the only point of Proofreading in
Translation. It is responsible for Fidelity/Accuracy of Protein Synthesis.
z Amino Acyl tRNA Synthetase has 20 Isoenzymes, one for each Amino Acid.
80 : Molecular
z Hydroxy-Proline a derived Amino Acid does not require Amino Acyl tRNA
Synthetase.
z Initiation Codon is AUG which Codes for Methionine in Eukaryotes and N-Formyl
Methionine in Prokaryotes.
Factors in translation in prokaryotes & eukaryotes

Prokaryotes Eukaryotes
Initiation IF 1, 2, 3 eIF 1, 2, 3, 4F
Elongation EF-Tu, Ts, G eEF 1α, 1β, 1γ, eEF 2
Termination RF 1, 2, 3 eRF
IF - Initiation factor
eIF - eukaryotic Initiation factor
EF - Elongation factor
eEF - eukaryotic Elongation factor
RF - Releasing factor
eRF - eukaryotic Releasing factor
z For Initiation in Eukaryotes, ATP and GTP are required.

z For initiation in Prokaryotes, no ATP and GTP are required and Initiation Factors
IF-1, IF-2 and IF-3 are required.
z Shine Dalgarno Sequence (SD sequence) is present in Prokaryotes on mRNA which
is Purine rich and has complementary bases with 16s rRNA, present 10 base pairs
Upstream to the Initiation Codon.
z P- site/Polypeptide Site : always lies on left side and Polypeptide is released from
this site.
z A- site/Acceptor Site : All Amino Acids are accepted here EXCEPT first Methionine.
z In both Prokaryotes and Eukaryotes, N- terminal Methionine is removed before
Translation is completed.
z Peptidyl Transferase releases Polypeptide from P-site. It has Role in Elongation and
Termination.
z Addition of Amino Acid to the Carboxy end of growing chain is known as Elongation.
z Small ribosomal unit binds mRNA and determines the accuracy of Translation by
correct Base Pairing.
z Large Ribosomal Unit catalyze formation of Peptide Bond.
z Releasing factors (RF1, RF2, RF3) recognizes Stop Codons in Prokaryotes.
z GTP is required for Termination in both Eukaryotes and Prokaryotes.
z 2 ATP (activation of Amino Acid) and 2 GTPs (one GTP for entry of Amino Acid
at A-site and other for Translocation) are required to add one Amino Acid in a
growing polypeptide chain.
z Both Transcription and Translation occur in Cytoplasm in Prokaryotes.
z In Eukaryotes, Transcription occurs in Nucleus and Translation in Cytoplasm and
hence a discontinuous process.
z Co-Translational Modification occurs in case of Selenocysteine and Pyrrolysine.
z Streptomycin binds 30S-50S interface and inhibit Initiation.
z Chloramphenicol inhibits Peptidyl Transferase.
Molecular : 81
z Tetracyclin binds to 30S subunit of Ribosome.

z Erythromycin inhibits Translocation.
z Dipthera toxin inhibits EF-G and EF-2.
z Post Translational Modifications e.g. Covalent Modification → phosphorylation,
Glycosylation, Hydroxylation, Acetylation, Vit K Dependent Carboxylation of
Glutamate, Biotin covalently bound to Carboxylases.
z Glycosylation is the most frequent Post Translational Modification of Protein.
z Non-Enzymatic attachment of Sugar to Protein is known as Glycation.
TECHNIQUES IN MOLECULAR BIOLOGY

z Samples taken for Prenatal Diagnosis:
1) Amniotic fluid : known as Amniocentesis – done after 16 weeks of
Gestation.
2) CVS (Chorionic Villus Sampling) – done after 11 weeks of Gestation.
3) Fetal Blood Sampling : done after 18 weeks of Gestation.
z Adult samples which can be taken for DNA testing – Blood (WBCs), Cheek Swab,
Hair.
z Epigenetics is information in the Genome which alter the pattern of Gene Expression.
This is Chemical Modification of DNA or Proteins but is not change in DNA Code.
z Epigenetic Changes are transferred to next generation and are reversible.
z Epigenetic Changes are : DNA Methylation, PTMs of Histones (occurs mostly on
lysine residue on proteins) and changes in high order of chromosome structure.
z DNA Methylation of Cytosine of CG sites by Enzyme – DNA Methyl transferases

(DNMTs).
z Acetylation of Histone by enzyme HAT (Histone Acetyl Transferase).
z Deacetylation of Histone by enzyme HDAC (Histone Deacyetylases).
z Histone Methylation can be done by Histone Methyl Transferases.
z Epigenetic changes causes diseases such as Cancer, Aging and Fragile X-syndrome.
z Fragile-X-Syndrome is the expansion of the CGG Triplet Repeat in FMR-1 Gene
and therefore Gene Silencing (Promoter Site Methylation) followed by lack FMRP
Expression and Fragile X Mental Retardation.
z FMR-1 Gene (Fragile X Mental Retardation Gene) is present on long arm of
X-chromosome, which Codes for Fragile X- Mental Retardation Protein which is
required for the development of connection between Neurons.
z Genetic Anticipation : The number of repeats increases from generation to
generation until affected individual is produced.
82 : Molecular
z Epigenetic Changes regulate Gene Expression, inactivate X-chromosome and has a

role in Genetic Imprinting and Defence Mechanism.
z Euchromatin is transcriptionally active.
z Heterochromatin is transcriptionally inactive.
z Facultative Heterochromatin means the Chromatin has ability to become
transcriptionally active again. E.g. X-chromosome Inactivation in Female Mammals.
z Constitutive Heterochromatin is always inactive and repetitive and usually present
in Centromere & Telomere and provides structural role.
z Genomic Imprinting is an Epigenetic Phenomenon which results in Mono-Allelic
Expression i.e. allele from one parent is expressed (either Maternal or Paternal).
z Paternal Imprinting : means Paternal Allele is inhibited and only Maternal Allele
is functioning.
z Maternal Imprinting : means Maternal Allele is inhibited and only Paternal Allele
is functioning.
z Most DNA Methylation is removed at Fertilization and re-established during
Embryogenesis.
z Mechanisms of Genomic Imprinting: DNA Methylation at CG sites, Histone
Acetylation and Histone Deacetylation.
z Detection of GI: Na Bisulphite Method, ChIP, ChIP-Sequencing, Flourescent In
Situ Hybridization and Methylation-Sensitive Restriction Enzymes.
z Na Bisulphite Method detects DNA methylation.
z X-Chromosome Inactivation in females is correlated with extensive CG Island
Methylation.
z Alterations in Gene and CG Island Methylation Patterns are seen in Aging and in
Cancer.
z Chromatin Immunoprecipitation (ChIP) detects Protein-DNA Interactions and
Post-Translational Modifications of Histones.
z chip is Microarray, but ChIP is Chromatin Immunoprecipitation.
z ChIP-on-chip/ChIP-chip Technique combines Chromatin Immunoprecipitation
with DNA Microarray.
z ChIP-chip can detect Protein-DNA interactions in vivo.
z RNA Immunoprecipitation (RIP) and Cross-Linking Immunoprecipitation (CLIP)
detects binding of Protein to RNA.
z RIP uses Formaldehyde-Cross Linking to induce covalent attachment of Proteins
to RNA.
z CLIP uses UV-Cross Linking to induce covalent attachment of Proteins to RNA.
z UV-Cross Linking is irreversible and more specific i.e bind RNA with Proteins that
are in close proximity.
z Components of PCR (Polymerase Chain Reaction): ds-DNA, two Primers,
Thermostable Polymerase - e.g. Taq Polymerase, Deoxy-Ribonucleotides and Mg++.
z PCR involves Denaturation (Strand Separation), Annealing (Primer Addition) and
Elongation (Synthesis). These cycles of Denaturation, Annealing and Elongation are
repeated again and again to synthesize large amount of DNA.
Molecular : 83
z Thermostable DNA Polymerase enzymes with Proofreading function in PCR: Pfu

(Pyrococcus furious), Pwo (Pyrococcus woesei) Polymerase and Tli (Thermococcus
litoralis)/Vent Polymerase.
z Best thermostable DNA Polymerase enzyme with Proofreading function is Pfu
Polymerase.
z Pfu is used in conjunction with Taq Polymerase to obtain fidelity of pfu with the
speed of Taq Polymerase activity.
z Real time PCR is also known as Quantitative Flourescence PCR(QF-PCR)/ qPCR.
z Real time PCR quantitatively measures the Amplification of DNA.
z SYBR Based Detection: Uses SYBR Green dye (dsDNA binding dye).
z TaqMan Based Detection: Uses a Florogenic Probe specific to Target Gene to detect
target as it accumulates during PCR.
z Reverse Transcription PCR/ RT-PCR is used to detect RNA Expression. In this,
sample RNA is converted to cDNA by enzyme Reverse Transcriptase.
z Tth Polymerase (Thermus thermophilus HB-8) has Reverse Transcriptase activity
when mixed with Manganese ions and thus can be used for the Amplification of
RNA to cDNA. It also has Polymerase activity.
z Quantitative Flourescent PCR (QF-PCR) use STRs to detect Aneuploidy.
z PCR is specific, simple, sensitive, flexible, rapid and relatively inexpensive technique
which amplifies DNA from low quantities and can work on damaged DNA.
z PCR has many applications such as Structural Analysis, DNA Typing, Disease
Detection, Cloning, Detection of Gene Expression, Mapping, Site Directed
Mutagenesis and Sequencing.
z Ligase Chain Reaction (LCR) or Ligation Amplification Reaction (LAR) is a method
for Amplification of DNA in which Probe is amplified.
z In LCR, enzyme used is thermostable DNA Polymerase and thermostable DNA
Ligase (Taq DNA Polymerase and Taq DNA Ligase).
z Multiplex Ligation-Dependent Probe Amplification (MPLA) is a kind of multiplex
PCR technique that can simultaneously analyze multiple Genomic regions.
z MLPA has small sample requirement.
z MLPA can detect copy number variations and can detect deletions and duplications
of any size.
z Karyotyping is the best technique for Monosomy/Trisomy but can be done only
during Metaphase of Chromosome.
z Karyotyping cannot be done in any phase of cell cycle. It cannot Detect Amplifications,
Micro-deletions and Complex Translocations.
z FISH (Fluorescent In-Situ Hybridization) is a rapid/fast technique which can be
done in any phase of cell cycle.
z FISH can be done in morphologically intact cell/tissue/organ.
z FISH can detect Monosomy/Trisomy, Micro-deletions, Amplifications and
Complex Translocations.
z FISH tells Gene location on a Chromosome.
84 : Molecular
z Restriction Fragment Length Polymorphism (RFLP) is a lengthy procedure and can

detect only single mutation and those mutations which are affected by Pallindrome.
z Probe is a DNA sequence, which is complementary to Gene of Interest or only a
small portion on Gene of Interest.
z Probe is labelled with a Radioactive material or a Fluorescent Dye.
z Hybridization/Blotting is used for visualization of sample DNA.
Technique Sample Analyzed Probe Gel Used

Southern Blot/ DNA DNA Yes
DNA-DNA
Hybridization
Northern Blot/ RNA DNA Yes
RNA-DNA
Hybridization
Western Blot/ Protein Antibody Yes
Immune Blot
South Western Blot Protein-DNA DNA No
Microarray mRNA or cDNA DNA No
ELISA Protein or Specific Antibody No
Antibody for Protein to be
detected
z Single Gene Expression Analysis can be done by both Northern and Western
Blotting.
z In Microarray/chip, small DNA fragments or Probes are immobilized on a solid
support e.g. glass slide, in an ordered fashion.
z Microarray can detect Multiple Mutations, Multiple Gene Expression Analysis,
Global Pattern of Gene Expression, SNPs, Genetic Transfer of Disease.
z Microarray cannot detect Aneuploidy i.e Monosomy/Trisomy.
z Comparative Genomic Hybridization (CGH) or Chromosomal Microarray
Analysis (CMA) is a cytogenetic method for analysis of copy differences between
two genomes.
z If hybridization is done on Microarray plate (instead of using metaphase
chromosomes) then is known as Microarray based CGH.
z CRISPR – Clustered Regularly Interspersed Short Pallindromic Repeats.
z Cas → CRISPR Associated Proteins.
z Cas 9 → an Endonuclease.
z CRISPR-Cas9 System is known as RNA Guided Nuclease system.
z CRISPR-Cas9 System is in Prokaryotes, to destroy Bacteriophage DNA.
z CRISPR and Cas Endonuclease acts together to degrade the Target DNA.
z CRISPR-Cas 9 System is used to know the Function of a Gene, for creating Double
Strand as well as Single Strand Breaks, Genome Editing, Gene Additions, Creating
SNPs and Altering Gene Transcription and Regulation.
Molecular : 85
z Conventional Karyotyping, FISH and CGH are cytogenetic techniques for Detection
of Mutations.
z Down Syndrome : 3 copies of Chromosome 21
z Edwards Syndrome : 3 copies of Chromosome 18
z Patau Syndrome : 3 copies of Chromosome 13
z FISH, RT-PCR, QF-PCR, MPLA are the methods for the detection of Specific
Chromosomal Aneuploidy.
z Sperm Sorting (Microsort Method) – separate X-bearing (larger in size & has
more DNA) & Y-bearing sperms to be used for Artificial Insemination or In-Vitro
Fertilization.
z Sanger’s Reagent is 1-Fluoro 2,4 Dinitro Benzene, used in DNA Sequencing.
z Hybridomas are cells which are engineered to produce a specific Antibody in huge
numbers, or used as source of DNA or RNA for Gene Cloning, Sequencing or Gene
Manipulations.
RNA INTERFERENCE
z RNA Interference/Gene Knock Down/Silencing Technique is a biological
phenomenon by which RNA molecules inhibit Translation.
z Gene Knock Down - means Gene is present but its function is suppressed.
z Gene Knock Out - means Gene is deleted.
z Micro RNA binds to 3’ end of target mRNA to inhibit Gene Expression.
z Drosha works with DGCR8 , also known as PASHA. It cuts primary micro RNA to
form pre-micro RNA.
z Dicer cut pre-microRNA to form dsRNA (around 20 Nucleotides in length).
z ss micro RNA combines with Proteins to form RISC (RNA-induced Silencing
Complex).
z RISC can bind to the target mRNA and can just suppress it or degrade it by enzyme
Slicer/Argonaute/Ago.
z siRNA (Silencing or small interfering RNA) is synthesized from cytoplasmic RNA
(e.g. tRNA or viral RNA) and can bind anywhere on the target mRNA.
nnn
CHAPTER
MISCELLANEOUS 8
z Ribosomes are organelles but they are Amembranous.

z Nucleus is rich in rRNA and it disappears during cell division.
z Mitochondria have their own mitochondrial DNA, RNA and Ribosomes which
synthesize around 10% of the Mitochondrial Proteins.
z Lipofuscin/Residual Bodies are formed when indigestible material gets accumulated
in lysosomes.
z Pompe’s disease is the only Glycogen Storage Disease which is a Lysosomal Storage
Disease. Rest all Glycogen Storage Disease affects Cytoplasm.
z Most severe PBD (Peroxisome Biogenesis Disorders) is Zellweger Syndrome
(Cerebro-Hepato-Renal Syndrome).
z Micrososmes do not exist in a cell. During cell fractionation, Rough ER is disrupted
to form Small Vesicles known as Micrososmes.
z Strongest covalent bond is Peptide Bond/Amide Bond.
z Weakest bond – Vanderwaals Interactions.
z Selenium deficiency can lead to Hypothyroidism.
z Three hormones involved in Homeostasis of Blood Calcium levels are Vitamin D,
Parathyroid Hormone & Calcitonin.
z Keshan’s Disease occurs due to Selenium Deficiency.
z Acrodermatitis enteropathica - Disorder of Zinc Metabolism.
Acrodermatitis enteropathica (moist erythematous plaques)
z Fluorosis occurs when Concentration of Fluorine is > 20 ppm.

z Vitamins which can cause Dementia - Vitamin B1, B3 and B12.
88 : Miscellaneous
z Nutritional causes of Cardiomyopathy are Deficiency of Thiamine, Selenium, Ca++,

Mg++ and Excess of Iron.
z Micronutrients in Glycolysis: Vitamin B1, B2, B3, Mg (for kinases).
z Minerals required in Energy - Mg++, Zn++, Chromium.
z Vitamins those are necessary for Neurological Function are Thiamine, Pyridoxine
and Cobalamine.
z Micronutrients required for Bones are Vitamin D (for Calcium Absorption),
Vitamin C (for Formation of Bone Matrix),Vitamin K, Mg++, Phosphate, Copper,
Zinc and Manganese.
z Factors which increase Iron Absorption : Vitamin C, Amino Acids containing –SH
group.
z Factors which decrease Iron Absorption : Antacids, Alkalies, Phosphates, Phytates.
z Fe+2 is required for Iron Absorption.
z Fe+3 is required for Iron Transport & Storage.
z Haem contains Fe+2.
z Factors which increase Calcium Absorption : Vitamin D, PTH (Parathyroid
Hormone), Lysine, Arginine.
z Phytates decrease Calcium Absorption.
z Vitamin K in its Coenzyme form is regenerated by enzyme Epoxide Reductase/
Vitamin K Reductase (required NADPH).
z Epoxide Reductase is inhibited by Oral Anti-Coagulants like Warfarin.
z Enzyme decreased in Lead Poisoning → ALA Dehydratase.
z Enzyme increased in Lead Poisoning → ALA Synthase.
z In Lead Poisoning, acid excreted in urine → δ-Amino Levulinic Acid.
z ALA-Synthase I Deficiency is Lethal.
z ALA-Synthase II deficiency is X-linked Sideroblastic Anemia.
z ALA-Synthase II is Rate Limiting Enzyme of Haem Synthesis in Erythroid Tissues.
z Most common Porphyria is Porphyria Cutanea Tarda (PCT).
z Every 3rd Amino Acid in Collagen is Glycine.
z Every 7th Amino Acid in Elastin is Valine.
z Most of Proline & Lysine of Collagen is converted to Hydroxy-Proline & Hydroxy-
Lysine.
z 3 enzymes required for Collagen Formation are Prolyl Hydroxylase, Lysyl
Hydroxylase and Lysyl Oxidase.
z Desmosine Cross Links are present in Elastin.
z Aldol Condensation (Intramolecular Cross Links) are present in Collagen.
z The most sensitive Acute Phase Reactant is CRP (C-Reactive Protein).
z Albumin is the most abundant Protein in Plasma.
z Albumin transports Most Steroids, T3, T4, Fat Soluble Hormones, Unconjugated
Bilirubin, Calcium, Magnesium and many drugs.
z Normal A/G ratio is 1.2-1.6.
z Ceruloplasmin is a Cu containing enzyme, which also has Ferroxidase Activity.
Miscellaneous : 89
z Ceruloplasmin transports Copper.

z Transferrin transports Iron.
z Transcortin transports Cortisol, Aldosterone & Progesterone.
z Haptoglobin transports Free Haemoglobin released from RBCs.
z Hemopexin transports Free Heme released from Haemoglobin.
z Haemosiderin and Ferritin are storage forms of Iron.
z Haemosiderin has higher Iron content than Ferritin.
z Hepcidin regulates Iron transport in circulation.
z Afamin transports Vitamin E.
z Alpha- Feto Protein transports Long Chain Fatty Acids & Bilirubin.
z Transthyretin transports Thyroxine & Retinol Binding Protein.
z OFR with Highest Activity (most powerful) is OH (hydroxyl).
z H2O2 is not a free radical but it can generate Free Radicals. Precursor of all ROS is
Superoxide Radical.
z MDA is not considered the Marker of Lipid Peroxidation.
z Nitric Oxide (Endothelium derived Relaxation Factor) is also a Free Radical.
z FOX Assay – (Ferrous oxidation in Xylenol) is for the measurement of Free Radicals.
z Chain Breaking Anti-Oxidants are α-Tocopherol, β-Carotene and Superoxide
Dismutase (SOD).
z Vitamins with Anti-Oxidant Activity: Vitamin A, C, E.
z Vitamin E is the most powerful Lipid Phase Antioxidant.
z Lipids are most susceptible for Free Radical Damage (known as Lipid Peroxidation).
z Enzymatic Antioxidants are Superoxide Dismutase, Catalase & Glutathione
Peroxidase.
z Diabetic situation is almost same like Fasting or Starvation → Increased β-oxidation
of Fatty Acids, Lipolysis, Proteolysis, Gluconeogenesis, Ketone Body Synthesis.
z In Diabetes: Snow Flake Cataract occurs.
z Alcohol has Zero order kinetics.
z No Negative Feedback Control for Alcohol Metabolism.
z Ethanol & Fomepizole – used in Methanol Poisoning.
z Alcohol/Ethanol causes Fatty Liver due to increased NADH/NAD Ratio.
z Protein Precicipation can be done by Heat, Strong Mineral Acids, Heavy Metal Salts
and Alkaloidal Reagents (Trichloro Acetic Acid, Sulfosalicylc Acid, Phosphotungstic
Acid), Organic Solvents (ether) and Neutral Salts (Ammonium Sulfate).
z Radio Immuno Assay (RIA) is used to assay Drugs, Vitamins, Hormones and to
analyze Anti-DNA Antibodies in SLE (Systemic Lupus Erythematosus).
z Most commonly used Method for HbA1C is Ion Exchange High Performance
Liquid Chromatography (Ion Exchange HPLC).
z Best Method for HbA1C is Enzymatic Assay (Horse-Radish Peroxidase enzyme
used).
90 : Miscellaneous
z Negative charge in Fibrinopeptide A & B is due to Acidic Amino Acids (Aspartate

& Glutamate).
z Bacteria in yoghurt produces a form of Lactase which breaks Lactose. So curd doesn’t
contain Lactose, so can be given to Lactose Intolerant Patients.
z Type IV Collagen (present in Basement Membrane of Glomerulus) is defective in
Alport Syndrome.
z Type VII Collagen (present at the junction of Dermis & Epidermis) is defective in
Epidermolysis Bullosa.
z Fish Odour Syndrome/Trimethyl Aminuria - a rare Metabolic Disorder due to
absence of enzyme Trimethyl Amine Oxidase (Flavin Monoxygenase), which requires
Vitamin B2. Sources of Trimethylamine are dietary Choline, Trimethylamine Oxide
by Bacteria, Egg Yolk, Liver, Fish. Trimethylamine is normally oxidized in Liver by
enzyme Trimethyl Amine Oxidase to Trimethyl Amine Oxide (odorless), which is
excreted in Urine. But if this enzyme is not there, then Trimethyl Amine is excreted
in Urine, which gives Rotten Fish like Odour to Saliva, Sweat & Urine. Restriction of
Choline Rich Foods (Fish, Eggs, Liver, Nuts, Grains).
z Chaperones (located in Rough Endoplasmic Reticulum) are used in Protein Folding
e.g. BIP (Immunoglobulin Heavy Chain Binding Protein), GRP-94 (Glucose
Regulated Protein), GRP-170, GRP-78, Calnexin, Calreticulin, PDI (Protein
Disulfide Isomerase), PPI (Peptidyl Prolyl Cis-Trans Isomerase), HSP-47, ERp29,
ERp57.
z Calbindin is a group of several Calcium Binding Proteins.
z Subacute Combined Degeneration of Spinal Cord (known as Lichtheim’s Disease)
– Degeneration of Posterior & Lateral Column- occurs most commonly due to
vitamin B12 Deficiency, but also can occur in vitamin E or Copper Deficiency.
z Most abundant Pro form of Vitamin A is Beta-Carotene, which gets broken down
to two molecules of Retinal.
z The Secretory Vesicle are vesicles Secretory Vesicle
that mediate the Vesicular
Transport of Substances - e.g.
Hormones or Neurotransmitters -
from an organelle to specific sites
by fusing with the Cell Membrane
to release its content.
Miscellaneous : 91
Substrate utilized for energy production

FED FASTING STARVATION
Brain Glucose Glucose Ketone bodies
Heart Fatty acids Fatty acids Ketone bodies
Liver Glucose Fatty acids Amino acids
Muscle Glucose Fatty acids Fatty acids & K.B.
Adipose tissue Glucose Fatty acids Fatty Acids
RBC Glucose Glucose Glucose
Cognomen
Name Other Name
Sarcosine N-Methyl Glycine
Betaine Trimethyl Glycine
Choline Trimethyl Ethanolamine
Carnosine β-Alanyl Histidine
Anserine Carnosine on methylation
Homo Carnosine GABA + Histidine
Melatonin Acetyl Methyl Serotonin
Serotonin 5-OH Tryptamine
Ethanolamine Serine on Decarboxylation
Β-Mercapto Ethanolamine Cysteine on Decarboxylation
Specialized Products from Amino Acids

Amino Acid Metabolic Products
Aspartate z N1 of Purine
z N1, C4, C5, C6 of Pyrimidine
z Urea Synthesis
Arginine z Nitric Oxide

z Agmatine
z Ornithine and Urea
z Creatine
Cysteine z Cystine
z Taurine
z Glutathione
z Beta mercapto ethanolamine
Glutamate z N-Acetyl Glutamate

z Glutathione
z γ-Amino Butyric Acid
92 : Miscellaneous
Amino Acid Metabolic Products

Glutamine z N3 and N9 of Purine
z N3 of Pyrimidine
Glycine z Purine
z Heme
z Glutathione
z Creatinine
Histidine z FIGLU
z Histamine
Tryptophan z Melanin
z Catecholamines (Epinephrine,
Norepinephrine, Dopamine)
z Thyroxine
Tyrosine z Serotonin
z Melatonin
z Niacin
Enzyme Code or Commision Numbers

Enzyme Enzyme Commision Number
z Any Kinase 2
z Thiokinase 6
z Succinate Thiokinase 6
z Any Phosphorylase 2
z Glycogen Phosphorylase 2
z Any Phosphatase 3
z Glucose-6-Phosphatase 3
z Any Mutase 5
z Any Carboxylase 6
z Any Dehydrogenase 1
z Glycogen Synthase 2
z Citrate Synthase 2
z ATP Synthase 3
z Nitric Oxide Synthase 1
z Aconitase (TCA) 4
z Fumarase (TCA) 4
z Enolase (Glycolysis) 4
z PEPCK (Gluconeogenesis) 4
Miscellaneous : 93
Biochemical Markers of Various Organelles

Nucleus DNA, DNA Polymerase
Mitochondria Succinate DH, Glutamate DH
Lysosomes Acid Phosphatase
Peroxisomes Catalase
ER Glucose-6-Phosphatase
Ribosomes RNA
GA Galactosyl transferase
Cytoplasm Lactate DH
Plasma Membrane Na-K ATPase, 5’ nucieotidase
Homophones in Biochemistry
z D and L z Structural Isomers (Enantiomers
z d and l z Optical Isomers (Dextro-rotatory and Levo-
rotatory)
z Racemic Mixture z Equal d + l present(optically inactive)
z Racemase Enzyme z Enzyme which converts D and L (name
Racemase is misnomer)
z Dextrin z Hydrolytic Product of Starch
z Dextran z Branched, HMW Homopolysaccharide made up
of α-Glucose (used as Plasma Volume Expander)
z Dextrose z Solution of Glucose
z Lactose z Made up of Galactose + Glucose (Present in

z Lactulose Milk)
z Made up of Galactose + Fructose (used as
Laxative)
z Lactate z Dead end of Glycolysis, Produced in Anaerobic
Glycolysis
z Lactase z Enzyme which breakdown Lactose
(Disaccharide)
z Lectin z Carbohydrate Binding Proteins, which play a role
in Recognition and Attachment
z Pectin z Polysaccharide, acting as a Soluble Dietary Fibre
z Lignin z A complex organic polymer, which is the major
Insoluble Dietary Fibre

z Insulin z Hormone, secreted from β-Islet cells of Pancreas
z Inulin z A Homopolysaccharide, made up of β-Fructose,
also Ideal substance for GFR
z Fructose 1,6 Bisphosphate z Involved in Glycolysis
z Fructose 1,6 Bisphosphatase z Involved in Gluconeogenesis
94 : Miscellaneous
z Fructose 2,6 Bisphosphate z Reciprocal regulator which increases the

rate of Glycolysis and decrease the rate of
Gluconeogenesis.
z Fructose 2,6 z Present in TIGAR (TP-53 Induced Glycolysis
Bisphosphatase and Apoptosis Regulator).

z Transferrin z Transports Iron
z Transcortin z Transports Cortisol, Aldosterone & Progesterone
z Transthyretin z Transports Thyroxine & Retinol Binding Protein
z Hemopexin z Transports Free Heme released from

z Haemosiderin Haemoglobin
z Hepcidin z Are storage forms of Iron
z Regulates Iron transport in circulation
Peculiar Odours in Different Amino Acidurias

Inborn error of Metabolism Urine Odour
Phenylketonuria (PKU) Mousy/Musty
Maple Syrup Urine Disease (MSUD) Maple Syrup
Isovaleric Acidemia Sweaty Feet/Cheesy
Hawkinsinuria Swimming Pool
Glutaricacidemia Sweaty Feet
3-Hydroxy-3-Methyl Glutaric Aciduria Cat Urine
Multiple Carboxylase Deficiency Tomcat Urine
Hypermethioninemia Boiled Cabbage
Tyrosinemia Boiled Cabbage, Rancid Butter
Trimethylaminuria Rotten fish
Mucopolysaccharidosis (MPS)
Disease Name Enzyme Deficiency Substrate Accumulated
I H-Hurler α-L-Idouronidase *DS+#HS
(Autosomal Recessive)
I S-Scheie α-L-Idouronidase *DS
II Hunter Iduronate Sulfatase *DS+#HS
(X-linked Recessive)
VI Maroteaux Lamy Aryl Sulfatase B *DS
*DS – Dermatan Sulfate, #HS – Heparan Sulfate

Miscellaneous : 95
Hyperlipoproteinemias
Type I/Familial Lipoprotein Lipase or z Chylomicrons
Hyperchylomicronemia Apo C-II z VLDL
(Autosomal Recessive) z TGs (Triglycerides)
Type II-α/Familial Absent or Defective z Cholesterol

Hypercholesterolemia LDL-Receptors or Apo z LDL
(Autosomal Dominant) B-100
Type II-β/Familial Unknown defect z VLDL
Hyperlipoproteinemia z LDL
z TGs
z Cholesterol
Type III/ Dysbeta- Apo E z Chylomicron remnant

Lipoproteinemia/ Broad z VLDL remnant/IDL
Beta Disease z TG
(Autosomal Recessive) z Cholesterol
Sphingolipidoses
Neimann-Pick Disease Sphingomyelinase Sphingomyelin
Gaucher’s Disease β-Glucosidase/ Glucosyl Ceramide
(Autosomal Recessive) Gluco-cerebrosidase
Krabbe’s Disease β-Galactosidase Galactosyl Ceramide
Fabry’s Disease α-Galactosidase Ceramide Trihexoside
(X-linked Recessive)
Farber’s Disease Ceraminidase Ceramide
Tay Sach’s Disease Hexosaminidase A GM2 Ganglioside
Sandhoff’s Disease Hexosaminidase A&B GM2 Ganglioside
Metachromatic Aryl Sulfatase A Cerebroside Sulfate
Leukodystrophy
GM1 Gangliosidosis β-Galactosidase
96 : Miscellaneous
Glycogen Storage Diseases (GSD)

Von-Gierke’s Disease Glucose-6-Phosphatase Glucose-6-Phosphate
Pompe’s Disease Acid Maltase Glycogen in Lysosomes
Cori’s Disease Debranching Enzyme Limit Dextrins
Anderson Disease Branching Enzyme Amylopectin
Mc Ardle’s Disease Muscle Phosphorylase Muscle Glycogen
Her’s Disease Liver Phosphorylase Liver Glycogen
Urea Cycle Disorders

Hyperammonemia Type I CPS-I (Carbamoyl NH3
Phosphate Synthetase-I)
Hyperammonemia Type II OTC (Ornithine NH3
Transcarbamoylase) OMP
UMP
Orotic Acid
Citrullinemia Type I Argino-Succinate NH3
Synthetase Citrulline
Arginosuccinic Aciduria Argino-Succinate Lyase NH3
Arginosuccinic Acid
Hyperargininemia Arginase NH3
Arginine
Citrullinemia Type II Citrin (Aspartate- Citrulline
Glutamate Transporter)
HHH Syndrome Ornithine transporter Hyperammonemia
(Autosomal Recessive) Hyperornithinemia
Homocitrullinuria
Miscellaneous : 97
Rate Limiting Enzymes

Pathway Rate Limiting Enzymes Regulators
Glycolysis Phosphofructokinase-I AMP ⊕, Fructose 2, 6
Bisphosphate ⊕
ATP , Citrate
TCA/Kreb’s Cycle/ 3 Enzymes depending ADP ⊕
Citric Acid cycle upon various conditions ATP , NADH
(Citrate Synthase, Isocitrate
Dehydrogenase, α-Keto
Glutarate Dehydrogenase)
Glycogenesis Glycogen Synthase Glucose-6-Phosphate ⊕,
Insulin ⊕
Epinephrine , Glucagon
Glycogenolysis Glycogen Phosphorylase Epinephrine ⊕, Glucagon ⊕,
AMP ⊕
Glucose-6-Phosphate ,
Insulin , ATP
Gluconeogenesis Pyruvate Carboxylase, Citrate ⊕
PEPCK, Fructose 1,6 AMP , Fructose 2,6
Bisphosphatase Bisphosphate
HMP Glucose-6-Phosphate NADP ⊕
Dehydrogenase NADPH
Fatty Acid Carnitine Acyl Transferase-I Malonyl CoA
Oxidation
Fatty Acid Acetyl CoA Carboxylase Insulin ⊕ , Citrate ⊕
Synthesis Glucagon , Palmitoyl CoA
Ketone Body HMG CoA Synthase Glucagon ⊕,
Synthesis Insulin , Malonyl CoA
Cholesterol HMG CoA Reductase Insulin ⊕, Thyroxine ⊕
Synthesis Glucagon , Cholesterol,
Mevalonate , Statins ,
Bile Acids
Purine Nucleotide PRPP Glutamyl Amido AMP , Inosine
Synthesis Transferase Monophophate(IMP) ,
GMP
Purine Catabolism/ Xanthine Oxidase -
Uric Acid Synthesis
98 : Miscellaneous
Pathway Rate Limiting Enzymes Regulators

Pyrimidine Carbamoyl Phosphate ATP ⊕ , PRPP ⊕
Synthesis Synthetase-II (CPS-II) UTP
Bile Acid Synthesis 7-α Hydroxylase N-Acetyl Glutamate ⊕
Urea Cycle Carbamoyl Phosphate NAG (N-acetyl glutamate) ⊕
Synthetase-I (CPS-I)
Porphyrin/Haem δ-Amino Levulinic Acid Haem
Synthesis Synthase (ALA Synthase)
Table of Vitamins
Name Active Form Function Deficiency Special Features
Vitamin B1/ z TPP z Oxidative z Beri-Beri z RBC
Thiamine (Thiamine decarboxylation z Wernicke- transketolase
Pyrophos- reactions Korsakoff activity to be
phate) z Transketolase Syndrome determined for
z Phosphorylation biochemical
of chloride assessment of
channel in brain deficiency
Vitamin B2/ z FMN z Electron transfer (Rare) z For detecting
Riboflavin/ z FAD z Riboflavinosis deficiency, RBC
Warburg z Dermatitis glutathione
Yellow z Angular reductase is
Enzyme Stomatitis assessed
z Cheliosis z Endogenously
synthesized by
bacterial flora
Vitamin B3/ z NAD+ z Electron transfer z Pellagra z Tryptophan
Niacin/ z NADP+ z (Diarrhea, synthesizes it
Nicotinic Dementia, z 60mg
Acid/ Dermatitis, tryptophan
Nicotinamide Death) synthesizes 1mg
of Niacin
Vitamin B5/ z Coenzyme z Acyl carrier (Rare) z Contains
Pantothenic A z TCA z Burning Feet β-Alanine in its
Acid z ACP z Haem synthesis Syndrome structure
z Present in
CoA and Acyl
Carrier Protein
(ACP)
Vitamin B6/ z PLP z Transamination (Rare) z Deficiency can
Pyridoxine/ (Pyridoxal z Simple z Neurological be induced by
Pyridoxam- Phosphate) Decarboxylation Manifestations Isoniazid
ine/ z Trans- z Epileptic
Pyridoxal Sulfuration convulsions in
infant
Miscellaneous : 99

Vitamin B7/ z Biocytin z Carboxylation (Rare) z Consumption
Vitamin H/ z (Biotin+ reactions z Leiher’s Disease of Raw Egg
Biotin Lysine) z Regulation of z Exfoliative White can
Cell Cycle Dermatitis induce Biotin
Deficiency
z Synthesized by
Intestinal Flora
Vitamin B9/ z Tetrahy- z Carrier of one z Megaloblastic z Histidine load
Folic Acid drofloic Carbon Unit Anemia test/ FIGLU
Acid (THF) z Synthesis of z Nueral tube defect z Excretion test
Methionine, done to find
Purine, out Latent Folic
Thymidine Acid deficiency
Monophosphate
Vitamin B12/ z Methyl-co- z Cofactor for z Hyperhomo- z Folate Trap due
Cobalamine balamine reactions: cysteinemia to Vitamin B12
z Deoxy- Methionine z Pernicious deficiency
adenosyl synthase Anemia z In pernicious
cobalamine Leucine z Megaloblastic anemia, B12
aminomutase Anemia is given IM or
Methyl- z Dementia oral
malonyl CoA z Spinal
mutase degeneration
Vitamin C/ z Ascorbic z Antioxidant– z Scurvy z Can cross

Ascorbic Acid Acid cofactor for placenta
hydroxylation
reaction:
Proline →
Hydroxy-
Proline
Lysine →
Hydroxy-
Lysine
Vitamin A/ z Retinol z Reproduction z Bitot’s spots z Excess Vitamin
Retinol/ z Retinal z Vision z Night blindness A is toxic, leads
Retinal/ z Retinoic z Growth and z Growth to fractures
Retinoic Acid/ Acid Differentiation retardation
β-Carotene z Gene Expression z Xerophthalmia
Vitamin D/ z 1, 25- dihy- z Calcium and z Rickets (children) z Excess is toxic

Cholecalcif- droxy- Phosphate z Osteomalacia
erol/ z Cholecal- Absorption and z (adults)
Ergocalciferol ciferol (Cal- Reabsorption
citriol)
100 : Miscellaneous

Vitamin E / z Any of the Antioxidant
z (Rare) z High doses
α-tocopherol several Antiatherogenic
z z RBC fragility of Vitamin
tocopherol Role leads to hemolytic E depress
derivatives anemia Coagulability
z Edema
z Nuerological
problems
z Hemolytic anemia
z Retinopathy
z Dry skin and hair
loss
Vitamin K/ z Menadione γ-Carboxylation
z z In new borns z Produced by
Menadione/ z Menaqui- of glutamate z Fat Malabsorption GIT Flora
Menaqui- none residue in
none/ Phyl- z Phylloqui- clotting and
loquinone none other protein
Table of Minerals
Mineral Major Functions Deficiency
Sodium z Na /K ATPase pump
+ +
z Hyponatremia-
z Major Extracellular Cation Excess Sodium loss
z Acid Base Balance Excessive water
z Regulation of osmotic pressure retention

z Nerve transmission
z Maintainence of blood viscosity
z Electrolyte and Water Balance
Potassium z Na+/K+ ATPase pump z Hypokalemia-

z Major Intracellular Cation Muscular weakness
z Electrolyte and Water Balance Irregular heart beat
z Regulation of pH of body fluids Tachycardia
z Maintainence of Neuromuscular Altered ECG pattern
excitability
z Cofactor for enzymes e.g.
Pyruvate Kinase
Chloride z Formation of HCl in stomach z Hypochloraemia-
z Regulation of osmotic pressure of Severe vomiting
extracellular fluids. Addison’s Disease
Respiratory Acidosis
Metabolic Alkalosis
Calcium z Calcification of bones and teeth z Rickets in Children

z Blood Clotting z Osteomalacia in Adults
z Decrease membrane Fluidity z Osteoporosis in elderly
z Capillary permeability and nerve
excitabilty
Miscellaneous : 101

Phosphorus z Mineralization of bones and teeth z Rickets
z Constituent of High energy z Osteomalacia
phosphates like ATP
z Constituent of Nucleic Acids
Magnessium z Constituent of bones and teeth z Neuromuscular weakness

z Cofactor for Kinases,
Phosphorylases, Carboxylases
z Improves Glucose Tolerance
Iron z Constituent of haem z Second most prevalent

z Component of oxido-reductase nutritional deficiency
enzymes z Microcytic, hypochromic
z Electron transfer reactions anaemia

Copper z Cofactor for Oxidases z Microcytic, hypochromic
z Fe absorption and its anaemia
incorporation in the heme z Menke’s Disease
z Component of several enzymes
or proteins involved in redox

reactions
Selenium z Antioxidant Function z Keshan’s Disease
z Its action is complementary to z (Endemic
Vitamin E cardiomyopathy)
z Cofactor for Deiodinase
z Plays role in Spermatogenesis
Zinc z Zinc fingers-Important z Poor Wound Healing

constituent of regulatory proteins z Growth Retardation
that control transcription z Infertility
z Cofactor for Carbonic Anhydrase, z Hypogonadism
Alcohol Dehydrogenase, Lactate z Acrodermatitis
Dehydrogenase, Carboxy enteropathica

peptidase, Alkaline Phosphatase
Chromium z Component of ‘Glucose Tolerance z Hyperglycemia
Factor’ z Impaired Glucose
z Facilitates action of Insulin Tolerance
z Regulation of Gene Expression
Molybdenum z Cofactor for Xanthine z Severe Neurological

oxidase,Sulfite Oxidase, Aldehyde Abnormalities
oxidase, Lipoprotein Lipase
Cobalt z Constituent of Vitamin B12 z Pernicious Anaemia
z Maturation of RBCs
102 : Miscellaneous

Manganese z Required for Glycoprotein and z Growth Retardation
Proteoglycan Synthesis z Bone Deformity
z Cofactor for mitochondrial SOD, z Sterlity
Pyruvate Carboxylase, Glutamine z Fatty Infiltration in
Synthesis Hepatocytes
Iodine z Constituent of Thyroid hormones z Cretinism
z Goiter
z Myxedema
Flouride z Formation of Bones and teeth z Dental caries

z Provides resistance to tooth z Osteoporosis
enamels to sustain acid attacks
z Potent inhibitor of activities of
certain enzymes
ENERGETICS
z Number of ATPs in Anaerobic Glycolysis = 2 ATPs/glucose.
z Number of ATPs in Aerobic Glycolysis = 7 ATPs/Glucose.
z ATPs formed by Substrate Level Phosphorylation in Glycolysis are 4 → ATPs.
z ATPs formed by Oxidative Phosphorylation (ETC) in Glycolysis are → 5 ATPs.
z Number of ATPs in RBCs are never more than 2 , but it can be less than 2 if it is RL
(Rapaport Leubering) shunt.
z Link reaction gives 5 ATPs (as 2 NADH produced) from two Pyruvate. From one
Glucose, two Pyruvate are obtained.
z One Glucose on complete breakdown or complete oxidation = 32.
z One Glucose in a Cancerous Cell gives 2 ATPs (Warburg’s effect).
z One Acetyl CoA = 10 ATPs.
z One Palmitic Acid (16 C) = 106.
z One Stearic Acid (18 C) = 120.
z 2 pyruvate to glucose → 4 ATP & 2 GTP used.
z 2 lactate to glucose → 4 ATP & 2 GTP used.
z 2 alanine to glucose → 4 ATP & 2 GTP used.
z No ATPs used in :
HMP.
Uronic acid pathway.
Alpha oxidation.
Oxidation of very long chain fatty acids.
RL shunt.
Synthesis of ketone body.
nnn
IMAGE BASED
104 : Image Based
Organelles of a cell
Different types of endocytosis
Types of cell membrane transport systems

Image Based : 105
Molisch Test Barfoed’s Test
Benedict’s Test
Blue solution Green / yellow ppt Orange red ppt Brick-red ppt
No sugar present Traces of sugar Moderate amount Large amount sugar
present sugar present present
Rothera’s test Iodine test
Milky plasma Seliwanoff’s test

106 : Image Based
Osazones of various carbohydrates
Needle shaped glucosazone crystals as Galactosazone crystals as viewed under

viewed under the microscope the microscope (rhombic plates)
Powder puff/hedge hog shaped crystals of Sun flower shaped maltosazone crystals
lactose as viewed under the microscope as viewed under the microscope
Snow flake cataract Oil drop cataract
Circumcorneal vascularization – Bitot’s spots –

Vitamin B2 deficiency Vitamin A deficiency
Image Based : 107
Kayser-fleischer rings in Wilson’s Self mutilation in Lysch

Menke’s disease
disease (copper deposited in eyes) Nyhann syndrome
Albinism
Gaucher’s cell
Reilly bodies
z Reilly bodies, also known as Alder-Reilly bodies are found in many mucopolysaccharidosis
e.g. Hurler disease & Maroteaux-Lamy syndrome. The picture shows a neutrophil with
highly lobulated nucleus. They sometimes resemble toxic granulations, but reilly bodies are
not related to infecti on and are not transient in nature. These are metachromatic granules,
surrounded by a clear zone. These are present in the cytoplasm of all leukocytes including
neutrophils.
108 : Image Based
Enzymes
Competative inhibitor Non-competative inhibitor
Vmax same Km increases Vmax decreases Km same
Michaelis menton graph (graph between velocity & substrate concentration)-

rectangular hyperbola
Ion exchange chromatography

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ONE - LINERS

Dr. Smily Pruthi Pahwa

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Dr. Smily Pruthi Pahwa

1. Concepts in Biochemistry ........................................................................ 1-04

z Low Insulin - Glucagon Ratio means Anabolic State.

z Three pathways which occur both in Mitochondria & Cytoplasm are

z If Oxygen is added → known as Oxidation.

z Reductase belongs to EC Number 1 and it requires NADPH.

z Atkin’s diet is low calorie, low carbohydrate diet.

z Examples of D-Amino Acids are: D-serine and D-Aspartate → found in Brain.

z Oxidation at C-1 gives Aldonic Acid.

z Malt contains β-Amylase which hydrolyses starch into maltose by sequential

z Most widely distributed GAG → Dermatan Sulfate.

z Also known as Embden Meyerhoff Pathway/EMP pathway.

z Pyruvate is the end product in Aerobic Glycolysis.

PYRUVATE Pyruvate Acetyl CoA

z Link reaction (Conversion of Pyruvate to Acetyl CoA) is a link between Glycolysis

z TCA is both Anabolic & Catabolic i.e. it is Amphibolic.

z Malate Shuttle is important

z Mitochondrial Malate Dehydrogenase (MDH) converts Malate to Oxaloacetate

NAD+ Glycerol-3P FAD

Cytosol Inner mitochondrial Mitochondrial

ATP ATP ATP

ETC/ ELECTRON TRANSPORT CHAIN

z Cytochrome c is the only member of ETC, not lying in IMM.

Mitochondria) (2) Oxaloacetate

z Malate cross IMM and is again converted to Oxaloacetate by Cytoplasmic Malate

z Glucose-6-Phosphatase is common enzyme in Gluconeogenesis and Glycogenolysis.

z During exercise, Anaerobic conditions prevail and as Fats cannot be metabolized

Minor glycogenolysis 2. Pompe’s

z Branching enzyme is also known as Amylo α (1 → 4) (1 → 6 ) transglycosylase or

z In Liver, the function of Glycogen is to

z Skeletal Muscle does not contribute Glucose to blood as Glucose-6-Phosphatase

z Both Glycogenesis and Glycogenolysis occurs in Cytoplasm.

MINOR PATHWAYS OF CARBOHYDRATES :- HMP, Uronic Acid Pathway, Fructose

HMP (Hexose Mono Phosphate Pathway)

z HMP- Hexose Mono Phosphate

z Occurs in Liver, Adipose Tissue, Lactating Mammary Glands, Adrenal Cortex,

z Marker for B6 deficiency – Transaminase activity.

Uronic Acid Pathway

z Galactose Metabolism occurs exclusively in Liver.

z Galactose Metabolism occurs in 3 steps: Phosphorylation, Synthesis of UDP-

z Fructose ingestion does not lead to Fructose-1-Phosphate

z Substrate for Aldolase B is Fructose-1 Phosphate.

z In Essential Fructosuria, Benedict’s test as well as Seliwanoff’s test is positive.

NADPH NADP+ NAD+ NADH

z Excess Sorbitol in Lens leads to Snow Flake Cataract.

z Holoenzyme (Active Enzyme) = Apoenzyme (protein part) + Non-protein part.

z If organic, then known as Coenzyme.

z Coenzyme act as a Hydrogen Carrier as well as Carrier of Moieties other than

z Examples of Serine Proteases are Chymotrypsin, Trypsin, Elastase, Thrombin,

CLASSIFICATION Distinguishing Features

z Oxidoreductase (EC Number 1) carries Oxidation-Reduction Reactions.

z Large Km means a Low Affinity i.e. high concentration of substrate is needed to

z Suicidal Inhibition is shown by special class of Inhibitors in which enzyme converts

Allosteric Regulation of Enzyme

z Most common Covalent Modification is Phosphorylation and Dephosphorylation.

z Other Covalent Enzyme Regulation by Covalent Modification

I: Phosphorylation, II: Adenylation, III: Uridylylation,

z Enzymes active in Phosphorylated state are Glycogen Phosphorylase, Phosphorylase

OH NH2 CH3 NH2

z Free Amino Acids may be L-form or D-form.

Frame Shift Mutation

Splice Site Mutation

Type II → Topo-Isomerases cut two strands.

DNA Gyrase is a counterpart of Topo-Isomerase-II in Prokaryotes. This enzyme