Biochemistry PDF
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ONE - LINERS BIOCHEMISTRY:
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Pahwa Dr. Smily Pruthi Pahwa National faculty of PG & FMGE coaching in biochemistry biochemistry MBBS (Sri G Guru uru Ram Das institute of medic medical al sciences & research, Amritsar, Punjab) Punjab) MD Biochemistry m edical college coll ege Bi ochemistry (Dayanand medical & hospital, Ludhiana, Punjab) Punjab) SRship Christian medical college, Ludhiana, Punjab Punjab
© Nov 2018 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. completion. Firstandforemost I thankth thankthee Almighty for thispricelesslifeandfor bestowing upon his blessings to t o help me carry carr y out out this work with lots of courage courage and sheer determination. determination. Thank doesn’t seem seem sufficient sufficien t for my loving husband Jaspreet Singh whose Than k you doesn’t invaluable invalua ble presence is constant constant source of myhappin myhappiness ess and has always endowed me with strength to face challenges in life. Without his relentless support, this book bo ok would would have never never been been materialized. 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 financ financial ial suppor supportt and inspiratio inspiration n throu throughout ghout my work and lifting me uphill this phase of life. life. Special and an d heartiest heartiest thanks to my in laws for all the support as my parents and encouraging me. me. This book would never have been complete without my son Divaahaan who who book. sacrificed his mother’s time and let me to compose this book. It is an extraordinary joy to thank my assistant Dimple Nagpal whose constructivee criticism, constructiv criti cism, deep interest and intellectual acumen were instrumental in accomplishing this book. book. I would like to thank Roffers Publications for their efforts and inputs and in providing providi ng me a platform platform in bringing out out thebook. thebook. Finally the credit goes to all my students, who have been the actual inspiration for the composition of this particular book. book.
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. subject. It is fo forr sure a standout amongst the most most troublesome troublesome subjec subjects ts 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. 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. 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. chapters. The highlights of each topic have been refined and fine tuned to stimulate the learning process among the students. Seve Several ral tables tables and figures are added for the the elementary 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 postgradua te medical biochemistry students. This book serves as a ready r eady made source for MCQ preparation. 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. you develop a deeper understanding of the subject, you can have an access to videos To help you through mobile app “Biochemistry “Biochemistry by by Dr. Smily Pruthi” Pruthi” in in goo google gle play store. store. Good luck for your coming examinations. Genuine doubts are always welcome. You can be in touch wit with h me for any doubt at a t my Facebook Face book group. group.
Dr.SmilyPruthiPahwa Pahwa M.D. (Biochemistry)
CONTENTS
1.
Concepts Conc epts in Bio Biochemis chemistry try ............ .......................... .......................... .......................... .......................... ................ .... 1-04 1-04
2.
Carbohy Carb ohydrate drate Chem Chemistry istry .................................... ....................... .......................... ........................... ................... ..... 05-10 05-10
3.
Carbohy Carb ohydrate drate Metab Metabolis olism m .................................. ..................... .......................... ........................... ................... ..... 11-28 11-28 4.
Enzymes
29-36 29-36
5.
Amino Acids
37-52 37-52
6.
Lipids
53-62 53-62
7.
Molecular
63-86 63-86
8.
Miscellaneous
87-102 87-102
Colour ur Imag Images es .............. ........................... .......................... ........................... .......................... ......................... ............. 103-108 103-108 Colo
CONCEPTS IN BIOCHEMISTRY
CHAPTER
1
CONCEPTS IN BIOCHEMISTRY
Fed state - When we eat food. Fasting state - In between meals, when we are not eating food (Lack of food for12-18
hours).
Starvation - Severe or complete lack of nutrients (Lack of food since 1-3 days).
Low Insulin - Glucagon Ratio means Catabolic State. High Insulin - Glucagon Ratio means Anabolic State. Cyclic AMP causes Phosphorylation. Insulin decreases cyclic AMP and Glucagon increases cyclic cyclic AMP. So Insulin causes Dephosphorylation & Glucagon causes Phosphorylation.
is activated by Insulin, But only in the capillary beds of Adipose
Lipoprotein Lipase tissues. Hormone Sensitive Lipase is inhibited by Insulin. Hormone sensitive lipase is activated by Glucagon and other Insulin Antagonist Hormones. In Fed state, Acetyl CoA is used for the synthesis of Fats or enters TCA. In Fasting state, Acetyl CoA has three fates – TCA or Ketone Body Synthesis or TCA
Gluconeogenesis.
Fats can never be converted to carbohydrates because Link Link reaction reaction (Pyruvate (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. Acetyl CoA activates the veryfirst step of Gluconeogenesis i.e. Pyruvate Carboxylase (converts Pyruvate to Oxaloacetate).
2 : Concepts in Biochemistry Three pathways which occur both in Mitochondria & Cytoplasm are Gluconeogenesis, Urea cycle & Haemsynthesis. Pathways which occur in Cytoplasm are : Glycolysis, Glycogenolysis, Glycogenesis, HMP Pathway, Fatty Acid Synthesis, Cholesterol Synthesis. Pathways which occur in Mitochondria are : TCA, ETC, Replication, Transcription
and Translation ofBody mitochondrial DNA, β-Oxidation of Fatty Acids, Ketone Body Synthesis, Ket one Ketone Utilization. Sources of Blood Glucose are : Food, Liver glycogen (for 12-18 hrs) & Gluconeogenesis. Main fuel for body in Fed state is Carbohydrates. Main fuel for body in Fasting state is Fats. Heart uses Fatty Acids in fed state. Fetal Heart uses Glucose. In Heart Failure, fuel for Heart is Glucose. Brain cannot use Fatty Acids as Fatty Acids cannot cross Blood Brain Barrier. Ketone Bodies are formed during starvation for vital organs – Heart & Brain.
ENZYME CLASSIFICATION CLASSIFICATION Enzyme Code or Commission Number Enzyme Code or Commission Number Enzyme Code or Commission Number Enzyme Code or Commission Number Enzyme Code or Commission Number Enzyme Code or Commission Number
If Oxygen is added If Hydrogen added If Electron added
1 is Oxidoreductase. 2 is Transferase. 3 is Hydrolase. 4 is Lyase. 5 is Isomerase. 6 is Ligase.
known as Oxidation. known as Reduction. known as Reduction.
To break a bond, water is added. Hydrolase category uses water to break a bond. To make a bond, b ond, energy from ATP is used. Ligase category uses ATP to make a bond. Lyase category can make a bond, or break a bond, BUT neither uses water nor ATP. Transferases are enzymes which transfer a group from one molecule to another. Molecular formula is changed when Transferasework. Isomerases are enzymes which interconvert various isomers into each other. Molecular formula is not changed when Isomerase work. Dehydrogenase means removal of Hydrogen means Oxidation. If Dehydrogenase enzyme and there is removal of CO 2, then it is known as Oxidative Decarboxylation. If Dehydrogenase enzyme and there is removal of NH 2 , then it is known as Oxidative
Deamination. Reductive Biosynthesis means that in Anabolic Pathway, Reductase enzyme is required.
3 Concepts in Biochemistry : 3
Reductase belong belongss to EC Number Number 1 and it it requires requires NADPH. NADPH is formed by HMP and two minor sources are Malic enzyme and Cytoplasmic Isocitrate Dehydrogenase. NADPH is usedin Reductive Biosynthesis i.e. mainlyfor 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). NAD & FAD are used in Catabolic Pathways. Coenzyme for Cytoplasmic Isocitrate Dehydrogenase is NADP. Coenzyme for Mitochondrial Isocitrate Dehydrogenase is NAD. Addition of CO2 is known as Carboxylation . Enzyme is Carboxylase . It requires ATP,, Biotin (Vitamin ATP ( Vitamin B7), CO 2 & Mg2+ ions. Removal of CO2 is known as Decarboxylation. This is of two types : Oxidative Decarboxylation & Simple Decarboxylation. Carboxylation requires vitamin B7 (Biotin). Simple Decarboxylation requires vitamin B6 (PLP-Pyridoxal phosphate). Oxidative Decarboxylation requires vitamin B1 (Thiamine).
Vitamin (vitamin Thiamine B1). us Base. Thymine isisaaPyrimidine Nitrogeno Ni trogenous If FAD is used in some reactions, means means Vitamin B2 is used. If NAD is used in some reactions, means Vitamin B3 is used. If CoA is used in some reactions, means Vitamin B5 is used. Dehydrogenases are named after the substrate which gets oxidized e.g. Pyruvate Dehydrogenase. Reductases are named after the substrate which gets reduced e.g. Ribonucleotide Reductase. ATP is used when Kinases work. Whenever ATP is used, then Mg isused to stabilize stabilize the outgoing outgoing phosphate. Manganese Mangan ese can also al so be used instead of Magnesium. For Substrate Level Phosphorylation, enzyme is always a Kinase. For Oxidative Decarboxylation, enzyme is always a Dehydrogenase. Few ATPs are derived via Substrate Level Phosphorylation. Most ATPs in cells is derived via Oxidative Phosphorylation. ++ + ++ Cofactor for kinases : Mg . But Pyruvate Kinase requires K more than Mg . Kinase transfer organic phosphate and belongs to Enzyme Code Number: 2. Phosphorylase transfer inorganic phosphate and belongs to Enzyme Code Number:2. Phosphatase belong belongss to Enzyme Enzyme Code Number: Number: 3 (Hydrolase), becausethisenzyme
removes phosphate by adding water. Excess Carbohydrates get converted to Fats in body. 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. VLDL transports Endogenous Fat from Liver to Peripheral Tissues. Chylomicrons transports Exogenous Fat from Intestine to Peripheral Tissues.
4 : Concepts in Biochemistry
Atkin’s diet is low calorie, low carbohydrate diet. Fructose is the Most Lipogenic Sugar. Thermogenic effect offood isalsoknown as Specific DynamicAction(SDA) isthe
amount of energy required to digest, absorb, transport & metabolize the food. This is maximum for Proteins .
CHAPTER CARBOHYDRATE CHEMISTRY
2
CARBOHYDRATE CHEMISTRY
Hydroxy group always gives polarity and has high tendency to bind Phosphate. Aldehyde group is always present at C-1. Keto group is always present at C-2. Molecular formula of Carbohydrates is(CH2O)n where n = number of Total Carbons. Total number of Isomers poss possible ible = 2n , where n = number of asymmetric carbons.
If all the four valancies of carbon are occupied by different atoms or groups of atoms, then the carbon is Asymmetric. If any two, three or four valancies of carbon are occupied by same atoms or groups of atoms, then the carbon is Symmetric.
case case of Carbohydrates (aldehyde or keto) is Symmetric, Functional But only incarbon Linear inconfiguration . In cyclic configuration functional carbon is
Asymmetric . Any compound having asymmetric carbon will show both Optical and Structural Isomerism. Carbohydrates and Amino Acids have asymmetric carbon so both Structural and Optical Isomerism present. Molecules which have Same molecular formula and Different structure are known as Structural Isomers. Molecules which which have Same molecular formula and Different optical properties propertie s are known as Optical Isomers. Structural Isomerism is of four types functional isomerism, enantiomerism, Epimerism and Anomerism.
Number of asymmetric asymmetric Carbons Carbons in Ketoses Ketoses are one less the number in aldoses. The only carbohydrate with zero asymmetric carbon is Di Hydroxy Acetone (DHA). Glycerol. Parent alcohol of Carbohydrates Glycerol. Parent carbohydrate D-Glyceraldehyde. Simplest carbohydrate D-Glyceraldehyde. Functional Isomerism - Functional group different (ald or keto). Enantiomerism - different -H and -OH orientation around the penultimate carbon. Enantiomerism, also known as -D and -L isomerism, also known as mirror images of each other. Enantiomer of carbohydrate abundant in Body/Nature/Plasma/Cell Always D form Or For Carbohydrates , it is alway al wayss ‘D’ form which is abundant. Enantiomer of Amino Acid abundant / abundant in Proteins/ synthesized in our body L form. Enantiomer of a Free Amino Acid in body Both D and L form exists.
6 : Carbohydrate chemistry Examples of D-Amino Acids are: D-serine and D-Aspartate found in Brain. Source of D-aminoacid is always exogenousi.e. fromdiet or fromintestinal bacterial flora. Epimerism - different -H and -OH orientation around only one carbon, other than the penultimate carbon. Mannose is an epimer of Glucose at C-2. Galactose is an epimer of Glucose at C-4. Mannose and Galactose are not epimers of each other, as they differ in two carbons. Anomerism - different -H and -OH orientation around the functional carbon, in cyclic configuration. Anomerism exist only in cyclic structures or when carbohydrate is present in Solution Form. In cyclic structures, functional carbon makes a bond with second last carbon. Pyranose is a 6 membered ring. Furanose is a 5 membered ring. Number of of carbons in Pyranose Ring = 5 (as one atom is oxygen). Number of of carbons carbons in Furanose Ring = 4 (as one atom atom is oxygen). Hexoses (6C sugars) can exist as both Pyranose and Furanose.
(5C sugars) can only exist as Furanose. Pentoses Furanoseringsare moreflexible thanpyranoserings,henceselectedascomponents of nucleicacids. That’s why NucleicAcids have ribose which always exists as Furanose. Two types of Anomers are Alpha (α) and Beta (β). α means at functional carbon -OH group is downwards. β means at functional carbon -OH group is upwards. In most naturally occurring biomolecules, N-glycos N-glycosidic idic linkages are in βconfiguration. 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). Solution of Glucose is known as Dextrose. Glucose is always Dextro-rotatory.
Fructose is always Levo-rotatory. D & L are Enantiomers (Structural Isomerism) but D & L are Optical Isomers ‘D’ means OH is towards right side, ‘L’ means OH is towards left side. ‘d’ means Dextro-rotatory and ‘l’ means Levo-rotatory . Racemic mixture is equal d & l (optical isomers). It is optically inactive. Racemase enzyme is that enzyme which interconverts D & L into each other (structural isomers). N-Acetyl Neuraminic Acid i.e. NANA (9 carbon sugar/ nanose) is also known as Sialic Acid. Apart from being an important constituent of Gangliosides, Sialic acid is known to act as Ageing marker for the protein in plasma. The proteins devoid of sialic acid are removed from the circulation very fast and hence have shorter half lives. Monosaccharides on oxidation forms Acids. Monosaccharides on reduction forms Alcohols .
Carbohydrate chemistry : 7 Oxidation at C-1 gives AldonicAcid. Oxidation at C-6 or oxidation at primary alcohol gives Uronic Acid. Oxidation at both C1 and C6 gives Saccharic Acid. Alcohols formed by reduction of Carbohydrates/ monosaccharides are hygroscopic in nature i.e. they absorb water. Type of Cataract in Diabetic patients Snow Flake Cataract. Type of Cataract in Galactosemia patients Oil Drop Cataract. Reducing disaccharides are - Lactose, Maltose, Isomaltose. Non-reducing disaccharides are - Sucrose and trehalose. Lactose(Milksugar) --1, 1, 4 glycosidiclinkage andyields D-galactoseand D-Glucose on hydrolysis. All monosaccharides are reducing. Molisch Test: general test for Carbohydrates. But one condition number of carbons should be 5 or more, only then Molisch’s Test is positive. Benedict’s Test: given positive by all reducing sugars. Benedict’s Testisa semi-quantitative test i.e. it gives anidea of thequantity of sugar present. Reducing agents such as Uric Acid, Ascorbic Acid, Levodopa, Methyldopa,
Homogentisic Acid, Salicylates can interfere in Urine Analysis testGlutathione andUristixand tests- Benedict’s . Barfoed’s Test: distinguish between Monosaccharides and Disaccharides. It is given positivee by Reducing Monosaccharide positiv Monosaccharides. s. Positive test test is red color. Monosaccharides Monosaccharides react very fast whereas Disaccharides react very slowly. On prolonged heating, Disaccharides can also give this testpositive. Seliwanoff’s Test: distinguish between aldehyde and keto sugar. It is given positive by keto hexose hexose sugars sugars e.g. Fructose. Bial’s test: is specific for pentoses (bluish color). Hexoses generally react to give green, red or brown products. Only reducing sugars forms Osazones. Osazones of Glucose (Glucosazone crystals), Fructose and Mannose are Needle Shaped or Broom Stick likeappearance.
Osazones of Galactose (Galactosazone crystals) are like Rhombic Plate. Osazones of Maltose (Maltosazone) are Sunflower Shaped. Osazones of Lactose have Powder puff or Hedge Hog appearance. Routinely used method for blood Glucose estimation-GOD-POD test. Iodine test: positiv positivee 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. Starch is the major carbohydrate in diet, which contains Glucose. Storage form of carbohydrate in animals in liver and muscle is Glycogen. Glycogen is more branched thanstarch. Starch is made up of 20% Amylose (unbranched) and 80% Amylopectin (branched). Amylase and Maltase breaks (1 4) bonds. Isomaltase breaks α (1 6) bonds.
8 : Carbohydrate chemistry
Malt contains β-Amylase which hydrolyses starch into maltose by sequential
removal of disaccharide units from the non-reducingends. Dextran is a complex, highly branched HMW (high molecular weight) homopolysaccharide made up of α -Glucose. It is a Storage poly polysaccharide saccharide in yeast yeast and bacteri ba cteria. a. Dextran is used as a Plasma Volume Expander in patients of Hypovolemic Shock. Commercial name of gel used in Gel Filtration Chromatography Chromatograph y is Sephadex. Biochemically it is dextran. Heteropolysaccharides and Cellulose areunbranched. 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. Humans lack enzyme Cellulase which breaks bonds bonds in cellulose, cellulose, hence cellulose cellulose acts as fibre indiet. Cellulose is not broken due to beta anomerism at C-1. Cellulose, Hemi-cellulose and Lignin are Insoluble Fibres andareexcreted in its original form from intestine. Pectin and Gums are Soluble Fibres and absorb water thus converting to gel form and then excreted. Soluble fibres are better than insoluble fibres to prevent constipation. Honey is rich in fructose. Inulin (a homopolysaccharide) is made up of β -fructose. Inulin acts as a Prebiotic and is an ideal substance for the measurement of GFR. Inulin is not broken due to beta anomerism at C-2. 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). Heteropolysaccharides are also known as GAGs (Glycosa-Amino-Glycans) as they contain tandem repeats of amino sugar and uronic acid. The only carbohydrate with amino group Heteropolysaccharide or GAGs GAGs (Glycosa amino glycans).
GAGs are slimy and slippery because of of negative negative charge given given by carboxy, carboxy, sulphate and acetate a cetate.. The blood group (A,B,O) substances are Glycosphingolipids . Oligosacchari Oligosaccharide de that is a part of Blood Blood Group ‘O’ substance takes part in the binding of H. Pylori Pylori to the gastricmucosa. Hyaluronic acid is the longest GAG. No sulphate in hyaluronic hyaluronic acid. acid. Heparin is more sulphated than Heparin sulphate and has highest negative charge. Heparin acts as an Anticoagulant . Heparin Sulphate if accumulated then it is responsible for Mental Retardation. Heparin sulphate is a GAG with role in Retinal Cell-Cell Attachment or anycell cell adhesion. Keratin Sulphate does not contain any Uronic acid. Keratin sulphate is responsible for Corneal Transparency. Cornea has mainly Keratin sulphate and some Chondroitin Sulfate.
9 Carbohydrate chemistry : 9 Most widely distributed GAG Dermatan Sulfate. GAG synthesized mainly by Arterial Smooth Muscle Cells - Dermatan Sulfate. Dermatan sulphate in excess is responsible for Atherosclerosis . Most abundant GAG is Chondroitin Sulfate. Major component of Cartilage - ChondroitinSulfate. D-Lyxose is a pentose sugar and a nd is a constituent of ‘Lyxoflavin’ isolated from human heart muscle. 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. Chitin - principal component of the hard exoskeletons of Arthropods, consists of β-1-4 linkage between N-Acetyl-Glucosamine residues. Lectins are Carbohydrate Binding Proteins, which play a role in recognition & attachment. Lectins (e.g. Ricin, Hemagglutinin, Cholera toxin) participate in Binding of Nitrogen Nitro gen Fixing Bacteria, Purification Purification of Glycoproteins Glycoproteins and also in Adherence Adherence of E.coli to Intestinal Epithelium. Polar Glucose cross the Lipid Bilayer Membrane through Glucose Transporters.
of Glucose is done Transport for the Entrapment of and Glucose inside the cells. Phosphorylation SGLT-1 is for Glucose and Galactose in Kidneys Intestine. SGLT-2 is only for Glucose Transport present in Kidneys. GLUT-1 is present in Brain, Placenta, Kidneys and also in RBCs. GLUT-3 is present in Brain, Placenta, Kidneys. GLUT-1 and GLUT-3 are active during Fasting state whereas GLUT-2 is active during Fed state. Hypoglycemic action of Insulin is via GLUT-4. Only GLUT-4 is Insulin Dependent as it recruits GLUT-4 on the membrane of
peripheral cells. peripheral cells. GLUT-1 is most abundant in RBCs. Major GLUT in Neurons isGLUT-3. Rate at which Glucose (solute) enters a cell via GLUT (Facilitative Transport) depends upon : 1) Number of of GLUT GLUTss 2) Concentration of Glucose 3) Affinity of GLUT G LUT for Glucose Glucose is the main energy source for Brain, RBCs, Retina, Cornea, Renal Medulla, Testis. RBCs can use only Glucose (in Fed/Fasting/Starvation). Glucose is the Universal Fuel for Fetus.
CARBOHYDRATE
CHAPTER
3
METABOLISM
GLYCOLYSIS Glucose Glucose
1
Hexokinase Hexokinase
ATP ADP ADP
Glucos Glucosee 6-phosphate 6-phosphate
2
Phosphoglucose Phosphoglucose isomerase isomerase
Fructose 6-phosphate
3
Phosphofructo- Phosphofructo- kinase-1 kinase-1
4
AldolaseAldolase-A A
ATP ADP ADP
1,6-bisphosphate Fructose 1,6-bisphosphate Triose Triose
Dihydroxyacetone phosphate phosphate
5 phosphate Glyceraldehyde Glyceraldehyde isomerase
6
3-phosphate (2 molecules) molecules)
Glyceraldehyde 3-phosphate dehydrogenase dehydrogenase
+
2 NAD + 2P1 + 2 NADH+ 2H
1,3-Bisphosphoglycerate (2 molecules) molecules)
7
Phosphoglycerate Phosphoglycerate kinase kinase
2 ADP 2 ATP
3-phosphoglycerate (2 molecules) molecules)
8
Phosphoglycero- Phosphoglycero- mutase mutase 2-phosphoglycerate (2 molecules) molecules)
9
Enolase Enolase Phosphoenolpyruvate (2 molecules) molecules)
10 10
Pyruvate kinase kinase
2 ADP 2 ATP
NADH NADH
NAD NAD Lactate (2 molecules) molecules)
Pyruvate Pyruvate (2 molecules) molecules)
11 11
Lactate Dehydrogenase (anaerobic) (anaerobic)
12 : Carbohydrate Metabolism Metabolism Also known as Embden Meyerhoff Pathway/EMP pathway. Glycolysis is activated by Insulin. Intracellular site for operation of Glycolysis : Cytosol. Occurs in all cells of the body. Occurs in both Aerobic and Anaerobic conditions. is the only molecule which can produce ATP without O . 2 Glucose Glycolysis is not the complete breakdown of Glucose. Glycolysis is irreversible. Steps 1 to 5 in Glycolysis constitute Phase I, also known as Energy Utilizing Phase as 2 ATPs areused. Steps 5 to 10 constitute Phase II of Glycolysis, also known as Energy Producing
Phase. Glucokinase/ Hexokinase IV is induced by Insulin. Glucokinase has specificity for D-Glucose. Hexokinase has Feedback Inhibition from the product Glucose-6-Phosphate. Hexokinase is Flux Generating Step of Glycolysis. 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 ATP NegativeEnzyme. Homotropic Regulator of PFK-1. is Limiting Citrate is Negative Heterotropic Allosteric Regulator of PFK-1. Inhibition of Glycolysis by Citrate directs Glucose for Glycogen Synthesis AMP & Fructose 2, 6 Bisphosphate are Positive Homotropic Allosteric Effectors
of PFK-1. Presence of ATP (indicates high energy state) inhibits PFK-1 and presence of ADP (indicates low energy state) activates PFK-1. Aldolase A belong belongss to Lyase categoryas it breaks Fructose 1, 6 Bisphosphateintotwo 3C molecules i.e. Glyceraldehyde-3-P and Dihydroxy Acetone Phosphate (DHAP) without using H2O. Aldolase B – a hepatic isoform is involved in Fructose Metabolism. a Glyceraldehyde-3-Phosphate Dehydrogenase is the only enzyme of Glycolysis which can use Inorganic Phosphate. Glyceraldehyde-3-P Dehydrogenase is inhibited by Iodoacetate and Arsenate. Conversion of Glyceraldehydes-3-P to 1, 3 Bisphospho Glycerate is the first Oxidation-Reduction step of Glycolysis. In Anaerobic Glycolysis, ATP is formed by Substrate Level Phosphorylation and not Oxidative Phosphorylation. Irreversible or Regulatory Steps of Glycolysis are Glucokinase, PFK-1 and
Pyruvate Kinase. Substrate Level Phosphorylation (SLP) steps of Glycolysis are Phospho Glycerate Kinase and Pyruvate Kinase. All SLP steps are reversible except Pyruvate Kinase which is irreversible. In Glycolysis, 7 steps are reversible , which are also used by Gluconeogenesis. MutaseusedinGlycolysis is Phospho-GlyceroMutase but Mutaseuse Mutaseusedin din Glycogen Glycogen Metabolism is Phosho-Gluco Mutase. Enolase is inhibited by Sodium Fluoride, which is used in Blood Glucose estimation.
Carbohydrate Metabolism : 13 13 Pyruvate is the end product in Aerobic Glycolysis. Excessive formation of Lactic Acid can be prevented by inhibiting PFK-1. Insulin activates Glucokinase, PFK-1 and Pyruvate Kinase. Pyruvate Kinase deficiency is the second most common Human Enzyme Deficiency. 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. Increased PFK-1 increases Fructose1,6 Bisphosphate which activates Pyruvate Kinase. Pyruvate Kinase is activated by AMP & Fructose 1,6 Bisphosphate. The extra step of Anaerobic Glycolysis occurs for the Replenishment of NAD . Lactate is the Dead End of Glycolysis. Lactate Dehydrogenase (LDH) is inhibited by Oxamate. Fates of Glucose-6-P are HMP, Glycogenesis and Gluconeogenesis Number of ATP produced produced in RBCs in Fed state, Fasting state, Aerobic state and Anaerobic state is 2 ATP. Rapaport Leubering (RL) Shunt occurs only in RBCs. Phosphoglycerate Kinase step is bypassed in RL Shunt. Number of ATPs in in RBCs are never more than than 2 , but it can be less less than than 2 if it is RL
(Rapaport Leubering) Shunt. PFK-I forms Fructose 1, 6 Bisphosphate. PFK-II forms Fructose 2, 6 Bisphosphate. Product of PFK-II ( Fructose 2,6 Bisphosphate) activates PFK-I. Fructose 2, 6 Bisphosphate is formed in Fed state & it activates Glycolysis but it
decreases Gluconeogenesis.
LINK REACTION Cytosol
Mitochondrion
T RANSPORT PROTEIN NAD+
(Pyruvate-Proton Symport) PYRUVATE
Pyruvate (3C) Coenzyme A
NADH
+ H
Pyruvate Dehydrogenase Complex
+
CO 2 Acetyl CoA (2C)
IRREVERSIBLE Oxidative Decarboxylation
5 coenzymes required are : Lipoic acid, Vitamin B1, B2, B3, B5 B5
Link reaction (Conversion of Pyruvate to Acetyl Acetyl CoA) CoA) is a link between betwee n Glycolysis & TCA. Occurs in Mitochondrial Matrix Link reaction step is Oxidative Decarboxylation (Requires Vitamin B1) Occurs in Fed state
Link reaction is activated byInsulin
14 : Carbohydrate Metabolism Metabolism Pyruvate enters Mitochondria via Pyruvate Proton Symport present in the Inner Inner Mitochondrial Membrane Link reaction enzyme is Pyruvate Dehydrogenase complex (PDH complex) PDH complex is a Multienzyme containing three components: 1) E1-Pyruvate Dehydrogenase or Pyruvate Carboxylase 2) E2-Dihydro Lipoyl Transacetylase 3) E3-Dihydro Lipoyl Dehydrogenas Dehydrogenasee PDH complex and TCA both requires five coenzymes – Vitamin B1, B2, B3, B5 and Vitamin
Lipoic acid Deficiency of these Vitamins (B1, B2, B3, B5) leads todecreased energyproduction and CNS problems as Brain cells depend upon this Aerobic reaction Allosteric Inhibition of PDH occurs by Acetyl CoA, NADH and ATP PDH is active in Dephosphorylated state and Dephosphorylation is done by Insulin Arsenate inhibits the enzymes requiring Lipoic Acid as coenzyme such as PDH complex Deficiency of PDH complex is the most common biochemical cause of Congenital
Lactic Acidosis A cidosis.. Beri-Beri causes Lactic acidosis
Link reaction is irreversible.
TCA CYCLE Acetyl CoA Oxaloacetate
Malate dehydrogenase dehydrogenase
Citrate synthase synthase
NADH NADH Malate
NA D
Citrate
Aconitase Aconitase
Fumarase Fumarase
Fumarate
Isocitrate FADH FADH
Succinate dehydrogenase dehydrogenase
NAD NAD
FAD FAD
NADH
Isocitrate Isocitrate dehydrogenase dehydrogenase
CO 2 Succinate
GTP
NAD NAD
GDP Succinyl hiokinase hiokinase
-ketoglutarate
NADH NADH -ketoglutarate dehydrogenase dehydrogenase
Succinyl-CoA
CO 2
TCA is both Anabolic & Catabolic i.e. it is Amphibolic. TCA is a Vital cycle, it has no Hormonal Control. TCA cannot occur in Anaerobic conditions and is strictly Aerobic . Occurs both in Fed as well as Fasting state. Neither activated activated by Insulin Insulin nor nor Glucag Glucagon. on.
Occurs in all cells of body where Mitochondria is present (occurs in Mitochondrial Matrix).
Carbohydrate Metabolism : 15 15 TCA known as Tricarboxylic Acid Cycle as Citrate and Isocitrate are Tricarboxylic Acids. Kreb’s cycle name is by name of scientist ‘Hans Adolf Krebs’. TCA is also called Citric Acid cycle as first compound formed is Citrate which is a Prochiral molecule. Prochiral Molecules are those which can be converted from Achiral to Chiral in a single step. Citrate inhibits Glycolysis and activates Fatty Acid Synthesis. Four steps involved in Oxidation-Reduction (Enzymes involved are Isocitrate Dehydrogenase, α-KG Dehydrogenase, Succinate Dehydrogenase and Malate Dehydrogenase. Decarboxylation tion (Isocitrate DHase and α -KG Two steps involved in Oxidative Decarboxyla DHase). Oxaloacetate (OAA) is regarded as First Substrate and Carrier of TCA and not Acetyl CoA. Oxaloacetate has a Catalytic role in TCA. Acetyl CoA is not the intermediate of TCA and is never Glucogenic. Aconitase always acts on the part of Citrate derived from Oxaloacetate.
is Non Competitive Inhibitor of Aconitase. Flouroacetate Flourocitrate is Competitive Inhibitor of Aconitase. Aconitase/Aconitate Hydratase belong belongss to Lyase Lyase category and is exceptiona exceptionally lly an
Iron-Sulphur Protein.
α-KG Dehydrogenase is a Multi Enzyme Complex similar to PDH complex and
requires same five coenzymes (TPP, Lipoic Acid, FAD, CoA, NAD) α-KG Dehydrogenase is not regulated by Phosphorylation or Dephosphorylation. Enzymes catalyzing Dehydrogenation without Decarboxylation do not require Metallic ions for their activity (Mg2+, Mn2+). 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 fo forr PEPCK enzyme enzyme of Gluconeogenesis.
There is only one Substrate Level Phosphorylation step in TCA. All enzymes of TCA lies in Mitochondrial Matrix Except Succinate Dehydrogenase (located in Inner Mitochondrial Membrane). Succinate Dehydrogenase Dehydrogenase isa is a Flavoprotein Enzyme in which FAD act as Hydrogen Acceptor and is inhibited byMalonate. Succinate Dehydrogenase is involved in TCA & ETC. Fumarate Hydratase/ Fumarase and Aconitase are Lyases. One Acetyl CoA gives 10 ATPs in TCAcycle (9 by Oxidative Phosphorylation/ETC and 1 by SLP). Anaplerotic Reactions are those reactions which replenish TCAintermediates. Most important Anaplerotic reaction is Pyruvate to Oxaloacetate conversion by
Pyruvate Carboxylase. Two CO2 in TCA are derived from the Carbons of Oxaloacetate. Irreversible steps of TCAare Citrate Synthase & α-Ketoglutarate Dehydrogenase.
16 : Carbohydrate Metabolism Metabolism
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
It
occurs in Cancerous Aerobic Glycolysis Cells. occurs in Cancerous cells but End Product is Lactate. One Glucose in Cancerous cells gives only 2 ATPs. Warburg’s effect is Aerobic Glycolysis and no Oxidative Phosphorylation.
CANCER CELL
Glycolysis
Pyruvate
Lactate
Oxygen
SHUTTLES Transport of NADH from Cytoplasm to Mitochondria occurs with the help of two
Shuttles namely Malate Shuttle and Glycerol Phosphate Shuttle. NADH cannot cross Inner Mitochondrial Membrane (IMM). So Shuttles are used to transport NADH from Cytoplasm to Mitochondria. NADH Shuttles (Malate Shuttle and Glycerol Phosphate Shuttle) are only used by those Pathways which occur in Cytoplasm.
Malate Shuttle is important in Liver and Heart.
Cytoplasmic Dehydrogenase
Malate
(MDH) converts Oxaloacetate to Malate and uses NADH.
Malate, Pyruvate & Aspartate can cross Inner Mitochondrial Membrane. Malate-Aspartate Shuttle
Mitochondrial Malate Dehydrogenase (MDH) converts Malate to Oxaloacetate
and produces NADH. Oxaloacetate cannot cross IMM. Malate Shuttle is also known as Malate-Aspartate Shuttle as Malate and aspartate cross IMM in this shuttle. Glycerol-P-shuttle is more important in Brain and Skeletal Muscles. Cytoplasmic Glycerol-3-PDehydrogenase enzyme converts Dihydroxy Acetone Phosphate (DHAP) to Glycerol-3-Phosphate and usesNADH.
Mitochondrial Glycerol-3-P dehydrogenase+ converts Glycerol-3-P to Dihydroxy Acetone Phosphate (DHAP) and uses FAD .
Carbohydrate Metabolism : 17
NA D
+
Mitochondrial glycerol-3-P DH
Cytoplasmic glycerol-3-P DH NADH+H
FAD
Glycerol-3P
+
DHAP
Cytosol
FADH
2
Inner mitochondrial Mitochondrial membrane matrix
Glycerol-3-Phosphate Shuttle
Malate Shuttle takes NADH from Cytoplasm and delivers NADH in the
Mitochondria. Glycerol Phosphate Shuttle takes NADH from Cytoplasm and delivers FADH 2 in the Mitochondria.
Glycerol
Phosphate Shuttle is a shorter shuttle and is a quick source of ATPs and is less prone to be affected by any deficiency.
Citrate Shuttle is used in Fatty Acid Synthesis. Citrate Shuttle transfers Acetyl CoA from Mitochondria to Cytoplasm . ATP Citrate Lyase breaks Citrate Citrate to Oxaloacetate Oxaloacetate and and Acetyl Acetyl CoA in Cytoplasm. Cytoplasm. Creatine-Phosphate Shuttle transport ATP from Mitochondria to Cytoplasm. ADP-ATP Translocase presen presentt in IMM IMM transfers transfers ATP ATP out into into intermembrane intermembrane
space and ADP transferred inside into the Mitochondrial Matrix. Enzyme Creatine Kinase – Mitochondrial (CKm) in Intermembrane Space converts Mitochondrial Creatine to Creatine Phosphate and this Creatine Phosphate reaches Cytoplasm.
Cytoplasmic Creatine Kinase again converts Creatine Phosphate to Creatine and
this phosphate is used to form ATP.
ATP
ATP
ATP
18 : Carbohydrate Metabolism Metabolism
ETC/ ELECTRON TRANSPORT CHAIN
NADH is the main starting material for ETC, also sometimes FADH . 2 NADH enters ETC and gives 2.5ATPs. FADH2 enters ETC and gives 1.5ATPs. ETC & TCA are Vital Pathways for cells and both occur in Mitochondria . ETC occurs in IMM, therefore, all components lie in IMM. ETC occurs in all cells of the body where Mitochondria is present. ETC occurs only in Aerobic conditions. ETC occurs in Fed and Fasting state. ETC has 5 Protein Complexes- Complex I, II, III, IV, V. Complex I to IV are involved in Electron Transport. Complex I is also known as NADH-CoQ Reductase / NADH Dehydrogenase. Complex II is also known as Succinate CoQ Reductase. Complex III is also known as Cyt C Reductase or CoQ-Cytochromec Reductase
or Cytochrome b/c1. Complex IV is also known as Cyt C Oxidase or Cytochrome a/a3 (Prosthetic
Group-Cu). Complex IV is the only Electron Carrier in which Haem Iron has an available coordination site that can directly react with O 2.
Complex V has twounits: 1) F0- present in IMM and is a Proton Channel. 2) F1- protrudes into Mitochondrial Matrix and is having enzyme activity of ATP
Synthase.
ATP Synthase is also known as F1/F0-ATPase because itit can also also catalyse catalyse the
hydrolysis of ATP to ADP and Pi.
CoenzymeQ (alsoknown asUbiquinone )istheonlyNon-Proteinmember of ETC. It is a Lipid Soluble Quinine and also an Anti-Oxidant.
Carbohydrate Metabolism : 19 19
Cytochrome c is the only member of ETC, not lying in IMM. Redox Potential is the tendency to gain electrons. has minimum Redox Potential (Strong Electron Donor). NADH O2 has maximum Redox Potential (Strong ElectronAcceptor ). Electrons Flow occurs from NADH to O2 in Electron Transport Chain.
Total 10 H+ are responsible for providing 2.5 ATPs from 1 molecule of NADH. + Complex I and Complex III transfers 4H , so they are responsible for the production of 1 ATP each. + Complex IV transfers 2H , so it is responsible for production of 0.5 ATP. When ETC starts from FADH2 , FADH2 gives electrons to Succinate Dehydrogenase, which further gives to Complex II and then to CoQ. Complex II does not form anyATP. In ETC, Oxidation and Phosphorylation are coupled i.e. they always occur together. Uncoupling means Oxidation is occurring but Phosphorylation does not occur. A substance which creates a hole in the IMM acts as an Uncoupler. Uncouplers – DNP (2,4 Dintrophenol), Thermogenin (known as Uncoupling protein-1/UCP-1) protein1/UCP-1),, Thyroxine and Free fatty acids.
Thermogeninisaproteinpresentin BrownFatandisresponsiblefor Non-Shivering Non-Shivering Thermogenesis . So Brown Fat is involved in Energy Expenditure ADPtoATPConversion or ComplexVisinhibitedbyOligomycin . It inhibitsboth
Oxidation and Phosphorylation. ADPtoATPtransfer is inhibitedbyAtractyloside. Enzyme inhibited is ADP-ATP Translocase. 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.
Rotenone and Phenobarbitone inhibits Complex I of ETC. Phenformin inhibits Complex III of ETC. Cyanide (CN), Carbon Monoxide (CO), Hydrogen Sulphide (H2S) inhibits
ETC.to Lactic Acidosis and Hyperuricemia. Complex IV ofleads Increased NADH
20 : Carbohydrate Metabolism Metabolism
GLUCONEOGENESIS
Occurs
in
Mitochondria Cytoplasm (starts
both and from
Mitochondria)
Organs: 90% in Liver (predominant) and 10% in
(2) Pyruvate Pyruvate 2 ATP Pyruvate carboxylase
2 ADP
(2) Oxaloacetate Oxaloacetate 2 GTP PEP Carboxy Kinase
2 GDP
Kidneys. Gluconeogenesis occur during Fasting/Starvation and in Diabetes Mellitus Firststepof Gluconeogenesis is conversion of Pyruvate to Oxaloacetate by Pyruvate Carboxylase enzyme (Anaplerotic reaction). Acetyl CoA is veryimportant Allosteric Activator of Pyruvate Carboxylase. Oxaloacetate produced in Mitochondria gets converted to Malateby Mitochondrial Malate Dehydrogenase as Oxaloacetate cannot cross IMM.
(2) PEP PEP
3-Phosphoglycerate (2) 3-Phosphoglycerate 2 ATP Phospho Glycerate Kinase 2 ADP (2) 1,3-Bisphosphoglycerate 1,3-Bisphosphoglycerate NADH + H
+
+
NAD
Fructose-1,6-bisphosphate P1
Fructose1,6 bisphosphatase
Fructose-6-phosphate
Glucose-6-phosphate P1
Glucose-6-Phosphatase Glucose Glucose
Malate cross IMM and is again converted to Oxaloacetate by Cytoplasmic Malate
Dehydrogenase. Phospho Enol Pyruvate Carboxy Kinase (PEPCK) converts Oxaloacetate to Phosphoenol Pyruvate which gets converted to Fructose1,6 Bisphosphate.
Enzymes common in Glycolysis and Gluconeogenesis : Enolase, Phosphoglycero
Mutase, Phospho Glycerate Kinase, Glyceraldehyde-3-P-Dehydrogenase and Aldolase A. All the bypass reactions of Gluconeogenesis are irreversible. Gluconeogenesis is not just the reversal of Glycolysis as three enzymes are different in Gluconeogenesis. Instead of Pyruvate Kinase in Glycolysis, Enzymes used in Gluconeogenesis are
Pyruvate Carboxylase and PEPCK. Instead ofPFK-1, Fructose 1,6 Bisphosphatase is usedin Gluconeogenesis Instead of Hexokinase, Glucose-6-Phosphatase is usedin Gluconeogenesis 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. Sequence of Compartments for Gluconeogenesis : Mitochondria Cytoplasm ER.
21 Carbohydrate Metabolism : 21
Glucose-6-Phosphatase is common enzyme in Gluconeogenesis and Glycogenolysis. Rate Limiting Enzymes for Gluconeogenesis are Pyruvate carboxylase, PEPCK and
Fructose 1,6 Bisphosphatase depending upon various complex situations in the body. body.
Substrates of Gluconeogenesis are Pyruvate, P yruvate, Lactate, Gly Glycerol cerol,, Propionic Acid, Acid,
Any TCA intermediate and Amino Acids Acids (Glucogenic category & both Glucogenic – Ketogenic Ketogenic category).
Purely Ketogenic Amino Acids are Leucine and Lysine (if both given mark Leucine
as Lysine is in controversy).
Both Glucogenic and Ketogenic Amino Acids Acids are Tyrosine, Tryptophan, Threonine, Isoleucine and a nd Phenylalanine Phenylal anine (Total Are Five) Five)..
Total Ketogenic Amino Acids = 7 (2 are purely Ketogenic and 5 are in both category). Total Glucogenic Amino Acids = 18 (13 are purely Glucogenic and 5 are in both category).
So only 2 Amino Acids can never form Glucose. Rest 18 can form Glucose Mostly 3-C containing compounds are Good Substrates for Gluconeogenesis e.g. Pyruvate, Lactate, Alanine. 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.
If Fructose 2,6 Bisphosphatase is active, Glycolysis decreases. Fructose 2,6 Bisphosphatase activity is present in TIGAR (TP-53 Induced Glycolysis & Apoptosis Regulator).
Two Pyruvate to Glucose formation by Gluconeogenesis uses 6 ATPs (or 4 ATPs & 2 GTPs). Two Lactate to Glucose formation by Gluconeogenesis uses 6 ATPs (or 4 ATPs & 2 GTPs). Two Alanine to Glucose formation by Gluconeogenesis uses 6 ATPs (or 4 ATPs & 2 GTPs). In Gluconeogenesis, ATPs are used by Pyruvate Carboxylase and Phospho Glycerol Kinase steps and GTP is used by PEPCK enzyme.
GLYCOGEN
Glycogen is the Storage form of Carbohydrates . Glucose is stored as Glycogen Glycogen because it packs a large quantity of Carbohydrates in small space, maintains low osmolarity in cells and interconvers inter conversion ion of Glucose Glucose and Glycogen provides a good
Regulatory Pathway.
The liver stores glucose as glycogen
Glycogenesis
Glycogenolysis
Blood Glucose
22 : Carbohydrate Metabolism Metabolism During exercise , Anaerobic conditions prevail and as Fats cannot be metabolized Anaerobically, only Glucose can be used which is obtained from Glycogen. Mobilization of Glycogen is faster than Fats. Fats can never be converted to Glucose so Fats cannot maintain Blood Glucose levels. Fatty acids cannot cross Blood Brain Barrier but Glucose can. Glycogenin is a Glycoprotein which which acts as a Primer in Glycogen synthesis Glycogenin also act as as Auto-Catalyst (in attaching Glucose on Glycogenin) OH group of Tyrosine in Glycogenin. OH Glucose gets attached to – Synthesis of Glycogen from Glucose is known as Glycogenesis. Glycogenesis is Anabolic Pathway and hence activated by Insulin and occurs in Fed state. cyclic AMP. AMP. Glycogen Synthase activity is depressed by cyclic In Glycogen, Straight chains have α (1 4) bonds and and the Branching point has α (1 6) bond. Two Glucose residues are joined by α (14) bond. Formation of UDP-Glucose is carried out by UDP-Glucose Pyrophosphorylase. Glycogen Synthase adds Glucose residues to the non-reducing end of Primer. Only straight chains are synthesized by Glycogen Synthase.
Minor glycogenolysis
2. Pompe’s
Acid Maltase Glycogen
Anderson 4. Anderson
5. Mc Ardle’s (Ms) (Ms) 6. Her’s (Liver) (Liver)
Branching enzyme
1->4 glucose units
1. Glycogen Phosphorylase (RLE (RLE)) 2. Glucan Transferase 3. Debranching enzyme
Glycogen Synthase RLE
UDP glucose UDP glucose pyrophosphorylase UTP PPi
Glucose-1-P Glucose-1-P Mutase
Glucose-6-P Glucose-6-P Glycogenesis
Hexokinase/ Glucokinase
Glucose-6-Pase
Glucose
3. Cori’s/Fobe’s/ Dextrinosis Limit Dextrinosis 1. Von Gierke’s
Glycogenolysis
Branching enzyme is also known as Amylo α (1 4) (1 6 ) transglycosylase or 4:6 transferase. The branched structure of Glycogen perm permits its the rapid rapid release release of Glucose Glucose simultaneously from every Non Reducing End of every branch. Breakdown of Glycogen to produce Glucose is called Glycogenolysis. Glycogenolysisis Catabolic Pathwayandhenceactivatedby Glucagon and occursin
Fasting state or in between meals. By weight, Glycogen is more in Liver (means when we take same weight of Liver & Muscle, then it is more in Liver). By percentage, Glycogen is more in Muscle (means when we calculate total body percentage Glycog Glycogen, en, then it is is more in in Muscle Muscle as Muscle Muscle Mass Mass is more). more).
Carbohydrate Metabolism : 23 23 In Liver, the function of Glycogen is to maintain Norma Normall Glucose levels.
End product of Liver Glycogenolysis is
free Glucose Glucose as Glucose-6-Phosphatase enzyme enzy me is present. Liver Glycogen stores st ores are increased in Fed state and depleted de pleted in Fasting. Fasting.
In Muscle, the function of Glycogen is to give energy for Muscle activity/ contraction.
Skeletal Muscle does not contribute Glucose to blood as Glucose-6-Phosphatase -6-Phosphatee. enzyme is absent. So, End product of Muscle Glycogenolysis is Glucose -6-Phosphat
This Glucose-6-P directly enters Glyco Glycolysis lysis.. Muscle Glycogen stores not affected in short period and are moderatelydecreased in prolonged Fasting. Minor pathway of Glycogenolysis occurs in Lysosomes (1-3%). Enzyme is Acid Maltase or Acidα (1 4)Glucosidase . Deficie Deficiencyof ncyof thisenzymeisknownas Type II Glycogen storage disease (Pompe’s disease). Pompe’s disease or Type II GSD is the only Glycogen Storage disease, which is a Lysosomal Storage Disease. Enzymes involvedinmajor pathway of Glycogenolysis are Glycogen Phosphorylase, Glucan Transferase and Debranching enzyme. Glycogen Phosphorylase (RLE) breaks α (1 4) bonds, starting starting from from non reducing reducing end, till four Glucose residues are left at the branch point. Glucose is liberated in the form of UDP-Glucose from Glycogen. Glycogen Phosphorylase (RLE of Glycogenolysis) is active in phosphorylated state. Activators of Glycogen Phosphorylase are cyclic AMP, Glucagon, Epinephrine/ Nor-Epinephrine NorEpinephrine,, Calcium- Calmodulin, Kinase and 5’AMP.
Epinephrine acts in Muscle and Liver but Glucagon acts only in Liver. Inhibitors of Glycogen Phosphorylase are Protein Phosphatase, Insulin, Glucose,
Glu-6-Phosphate, ATP and Fruct-6-Phosphate. 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 not releases releases any Glucose Glucose 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. Primary end product of Glycogenolysis obtained by breakin g α (1 4) bonds is
Glucose-1-Phosphate. Synthesis of Glycogenfrom Non Carbohydrate Sources isknownas Glyconeogenesis (Same as Gluconeogenesis except the last step of breakdown of Glucose-6-P not occurring and Glucose-6-P directly goes for Glycogenformation).
24 : Carbohydrate Metabolism Metabolism Both Glycogenesis and Glycogenolysis occurs in Cytoplasm. Glycogen Synthesis and Breakdown occurs occurs in Liver as well as in Muscle. Both Rate Limiting Enzymes of Glycogen Metabolism are Transferases. Free Glucose can cross Cell Membrane via GLUTs but Phosphorylated Glucose (Glucose-6-P is entrapped inside the cells. Enzyme common between Glycolysis and Glycogenesis – Hexokinase/Glucokinase. – Phospho-Gluco Enzyme common between Glycogenesis and Glycogenolysis
Mutase. Enzyme common between Glycogenolysis and Gluconeogenesis – Glucose-6-
Phosphatase. Most Glycogen storage disease (GSD) affect Glycogenolysis. Most common GSD – Type IGSD known as Von Gierke’s Disease and the enzyme Type deficient is Glucose-6-Phosphatase. Type I GSD clinical features are Severe Hypoglycaemia, Ketosis, Enlarged Liver and Kidneys, Hypertriglyceridemia, Lactic Acidosis and Hyperuricemia Causes of Hyperuricemia in Von Gierke’s Disease are Lactic Acidosis, HMP and Sequestration of Pi. Disease/Limit imit Dextrinosis and the enzyme deficient Type III GSD known as Cori’s Disease/L
is Debranching enzyme. Type IV GSD known as Anderson’s Disease/Amylopectinosis and the enzyme deficient is Branching enzyme. Type V GSD known as Mc Ardle’s Disease and the enzyme enzyme deficient is Muscle Phosphorylase. Type VI GSD known as Her’s Disease and the enzyme enzyme deficient is Liver Phosphorylase.
MINOR PATHWAYS OF CARBOHYDRATES :- HMP, Uronic Acid Pathway, Fructose Metabolism, Galactose Metabolism
HMP (Hexose Mono Phosphate Pathway)
HMP- Hexose Mono Phosphate Pathway because Glucose-6-P is the starting startin g material material HMP is also known as PPP (Pentose Phosphate Pathway) because Ribose-5-P Ribose-5-P is synthesized synthesized by this pathway pathway.. HMP is activated byInsulin. Intracellular site for HMP is
Glycolysis Glucose Glucose-6-Phosphate
Pentose Phosphate Pathway
Citric Acid Cycle
Cytosol. The only source of Ribose-5-P is HMP.
Electron Transport Chain
Occurs in Liver, Adipose Tissue, Lactating Mammary Glands, Adrenal Cortex, Gonads and RBCs.
Carbohydrate Metabolism : 25 25 HMP is the minor pathway pathwayfor for oxidation of Glucose but a major source for NADPH. NADPH produced in HMP is is needed for for Reductive Biosynthesis or to counter the damaging effects of Oxygen Radicals. No ATP is generated but CO2 is produced in HMP. HMP has 2 phases: Oxidative phase (irreversible) and Non-Oxidative phase (reversible). In cells with greater need for NADPH than Ribose-5-P, both Oxidative and NonOxidative Phase occurs. In cells with greater need for Ribose-5-P than NADPH, only Non-Oxidative Phase occurs. Oxidative Phase synthesize NADPH. Non-Oxidative Phase synthesize Ribose-5-Phosphate. Heptose keto sugar formed in HMP shunt is Sedoheptulose-7-P. 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. NADPH is a potent Competitive Inhibitor of Glucose-6-P dehydrogenase. Insulin upregulates the expression of gene for Glucose-6-P dehydrogenase
Pathways which do not produce ATP ATP are HMP, Uronic Uronic Acid Pathway Pathway and RL shunt, shunt, Synthesis of Ketone Bodies, α -Oxidation of Fatty Acids & Oxidation of VLCFA (Very Long Chain Fatty Acids). NADPH is produced produced from : HMP (major source), Malic enzyme and Cytosolic Isocitrate Dehydrogenase. In Oxidative Phase, 2 molecules of NADPH, 1 molecule of CO2 and 1 molecule of Ribulose-5-P is formed per Glucose-6-P moleculeoxidized. 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. ++ Transketolase requires TPP (derivative of Vitamin B1) and Mg . Transketolase transfers 2 carbon units. Transaldolase transfers 3 carbon units. Moleculewhichis Intermediate aswellasEndProduct in HMP- Glyceraldehyde-
3-Phosphate.
Moleculewhichis Substrate aswell as End Product in HMP- Glucose-6-Phosphate. Tissues which are never the site of HMP are Non-Lactating Mammary Glands and Skin. HMP activity is low in Skeletal Muscles and Brain (as in Brain, all the Glucose Glucose is used used by Glycolysis). Glycolysis). 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. Glutathione is a tripeptide (three Amino Acids are Glutamate, Cysteine and Glycine). It is a Reducing Agent (Reducing Property is due to SulThydryl group in Cysteine). Marker for B1 deficiency – Transketolase activity. Transketolase Marker for B2 deficiency – Glutathione Reductase activity. Glutathione
26 : Carbohydrate Metabolism Metabolism Marker for B6 deficiency – Transaminase Transaminase activity. G-6-PD deficiency is the first mostcommon Human Enzyme Deficiency. Pyruvate Kinase deficiency is the second most common Human Enzyme Deficiency. 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
Uronic Acid Pathway is also a Minor pathway for for the t he Oxidation of Glucose.
Uronic Acid Pathway synthesizes Glucuronic Acid, Acid, Pentoses and VitaminC. VitaminC. Humanscannotsynthesize Vitamin Cduetodeficiencyofenzyme :L-Gulonolactone Oxidase. Glucuronate is incorporated into Proteoglycans & it acts as Conjugating Agent (in Phase II conjugation reactions like Bilirubin Conjugation). Essential Pentosuria occurs due to deficiency of Xylitol Dehydrogenase or Reductase. Essential Pentosuria can also occur after consumption of large amounts of fruits that are rich in Pentoses.
L-Xylulose appears in urine in Essential Pentosuria which gives positive Benedict’s
Test (but Glucose Oxidase Strip test is negative). Garrod’s Tetrad (Pentosuria, Albinism, Alkaptonuria, Cystinuria).
Galactose Metabolism
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. cells.
Source of Galactose is Lactose (milk and milk products).. products) Galactose also obtained by Lysosomal
Glycoproteins and an d Glycolipids. Degradation of Glycoproteins Galactose Metabolism occurs exclusively in Liver.
Galactose ATP Mg2+
Galactokinase
ADP Galactose 1-phosphate
Block in galactosemia
UDPGIc
Galactose 1-phosphate uridyl transferase Glucose 1-phosphate
+
NAD
UDPGal
Uridine diphosphogalactose 4-epimerase
Carbohydrate Metabolism : 27 Galactose Metabolism occurs in 3 steps: Phosphorylation, Synthesis of UDPGalactose and Conversion of UDP-Galactose to UDP-Glucose. Galactokinase is responsible for the conversion of Galactose to Galactose-1-P. GALT (Galactose-1-P Uridyl Transferase) converts Galactose-1-P to UDPgalactose.
Conversion of UDP-Galactose to or UDP-Glucose is catalyzed by enzyme (UDP Galactose-4-Epimerase UDP-Hexose-4-Epimerase). This Epimerase reaction is
reversible. UDP-Galactose can be used for the synthesis of Glycolipids, Glycoproteins, GAGs and Lactose. Deficiency of Galactokinase is known as Minor Type Galactosemia. Excess Galactose gets converted to Galacitol/Dulcitol by enzym enzymee Aldose Aldose Reductase. Reductase. Cataract. t. Galacitol is hygroscopic in nature and in excess it causes Oil Drop Catarac 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. Confirmatory Diagnosis for GALT is Direct enzyme assay using Erythrocytes. Lactose synthesis in Mammary Glands occurs in Golgi by enzyme enzyme Lactose Synthase (also known as UDP-Galactose: Glucose Galactosyl Transferase) which transfers Galactose from UDP-Galactose to Glucose.
Fructose Metabolism
Fructose Metabolism takes place mainly in Liver. Fructose entry into cells is Insulin Dependent.
Fructose ATP ATP
Fructokinase Fructokinase ADP ADP
Fructose-1-Phosphate Fructose ingestion does not lead to Aldolase B (fructose-1-P Insulin release. aldolase) aldolase) Fructose is most Lipogenic sugar. Fructose gets rapidly metabolized as DHAP it bypasses PFK-1 step (rate limiting Glyceraldehyde ATP ATP step). kinase Triose kinase ADP ADP Fructose is more sweet than Glucose. Glyceraldehyde Substrate for Aldolase A and -3-Phosphate Aldolase C is Fructose 1,6 Enters Glycolysis Bisphosphate.
Substrate for Aldolase B is Fructose-1 Phosphate. Aldolase A is present in most tissues whereas Aldolase C is present in Brain. Energetics of Glucose and Fructose is same. So complete breakdown of Fructose =
32 ATPs. Unlike Glucokinase activity, Fructokinase activity is not affected by Insulin.
Fructose
metabolised normally in Diabetic Patient. Deficiency of Fructokinase is known as Essential Fructosuria/Benign Fructosuria.
28 : Carbohydrate Metabolism Metabolism In Essential Fructosuria, Benedict’s test as well as Seliwanoff’s test is positive. 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. Fructose is excreted in urine because of lack of renal threshold for Fructose.
is the deficiency of Aldolase B. Hereditary FructoseRecessive Intolerance HFI is an Autosomal (AR(HFI) ) disease.
HFI is usually asymptomatic but Fructose ingestion causes symptoms such as Hypoglycaemia, Jaundice, Lethargy, Hepatomegaly, Vomiting, Seizures and Irritability. HFI is treated by complete exclusion of Fructose, Sorbitol and Sucrose in the diet. Sorbitol Pathway synthesizes Fructose in body via Sorbitol.
Osmotic stress
Aldose reductase reductase
Glucose
N A D P H
GSSG
NADP
Glutathione reductase reductase
Sorbitol
+
Sorbitol dehydrogenase dehydrogenase
NA D
+
Fructose
NADH NADH
GSH
Excess Sorbitol in Lens leads to Snow Flake Cataract. Sorbitol Pathway leads to decreased NADPH and hence decreased Reduced Glutathione (antioxidant). Sorbitol in excess adds to Diabeticcomplications. Insulin Insensitive Tissues are Peripheral nerves, Renal glomeruli and Lens.
CHAPTER ENZYMES ENZYMES
4
ENZYMES
Enzymes are High Molecular Weight, Highly efficient and specialized Proteins,
which catalyze the Biochemical Reactions. Enzymes increase the Rate of the Reaction, without being changed in the overall process. All enzymes are Proteins except Ribozyme. Enzymes Enzy mes are Substrate Specific, Speci fic, Stereospecific and Thermolabile.
made of Binding Active Site istwo) Sites (usually & up Catalytic Site. Active site (A.S) is Flexible and not Rigid.
The A.S of enzyme is formed of Amino Acids which may be placed at long distances on the Primary Structure, but b ut are brought ne nearer arer to each other by 3-D Conformation of Enzyme.
Induced Fit Theory – Active Active Site is flexible, when substrate approaches active site
then there is conformational change which induces the active site to fit the substrate. For any Thermodynamically Favourable Reaction, the Free Energy of the Product is lower than that of Substrate. Amount of ΔG tells that a reaction is Thermodynamically Favourable or not. Negative ΔG (ΔG Mg ). ++ All Carboxylases need Mg . Cytoplasmic SOD require Copper but Mitochondrial Mitochondrial SOD requires requires Manganese. Isoenzymesarephysicallydistinct formofEnzymeswhichcatalyse sameBiochemical Reaction. Isoenzymes may be present in same/differenttissues.
Lactate Dehydrogenase (LDH) is a Tetramer made up of two subunits, H and M. LDH-1 (HHHH) is present in Heart. LDH-2 (HHHM) is present in Blood (WBC). So known as Functional Plasma Enzyme. In a Normal person, LDH-2>LDH-1. LDH-1 >LDH-2 in Myocardial Infarction (known as Flipped ratio of LDH ). Creatine Kinase (CK) has two subunits : B (Brain) and M (Muscle). CK-1 (BB) is present in Brain. CK-2 (MB) is present in Heart. CK-3 (MM) is present in Skeletal Muscles. LDH-1 and CK-1 has maximum Electrophoretic Mobility. LDH-5 and CK-3 has least Electrophoretic Mobility. CK-2 is raised in MI after 4-6 hours. AST/SGOT is raised in MI after 6-8 hours. LDH-1 is raised in MI after 8-10 hours. Earliest Marker for MI Myoglobin but it is Non Specific. Specific. Last Marker to rise in MI LDH. Most Specific Marker for MI = Troponin I > Troponin T. First Marker to fall in MI Myoglobin. Second Marker to fall in MI CK-MB. Last Marker to fall in MI LDH. IMA( Ischemia Modified Albumin) New Marker for Acute Myocardial Ischemia. Isoenzymes of Hexokinase are of four types (Type I, II, III, IV) and Type II being most abundant and overexpressed in Cancerous Cells. Hexokinase IV is known as Glucokinase. All Serine Proteases have Catalytic Triad (presence (pr esence of three Amino Acids at active site) of Histidine, Serine and Aspartate.
32 : Enzymes Enzymes Examples of Serine Proteases are Chymotrypsin, Trypsin, Elastase, Thrombin, Plasmin, Complements, Clotting Factors X and XI and PSA (Prostate Specific Antigen). Serine Proteases have role in Tumor Cell Metastasis . Chymotrypsin: cleaves BulkyHydrophobic AminoAcidse.g. Tryptophan, Tyrosine,
Phenylalanine. Trypsin : cleaves Basic Amino Acids e.g. Arginine, Lysine. Elastase : cleaves small Neutral Amino Acids e.g. Glycine, Alanine. Serine Proteases and Aminotransferases have Ping Pong Mechanism of Bi-Bi Reaction. Bi-Bi Reaction has two Substrates and two Products. Covalent catalysis often has Ping-Pong Mechanism (first substrate binds and
product released released and then the second substrat substratee will bind). bind). Oligonucleotide with single base change change is used used in Site directed Mutagenesis (not RFLP). Single Mutation is detected by RFLP (Restriction Fragment Length Polymorphism). Multiple Mutations are directed by Micro Array. CLASSIFICATION CLASSIFICATION
Distinguishing Features 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 Kinases
transfer amino groups 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 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
Oxidoreductase (EC Number 1) carries Oxidation-Reduction Reactions. Transferases (EC Number 2) catalyze Transfer of C-, N- or P groups e.g. Aminotransferase . Hydrolases (EC Number 3) uses water to break a bond. Lyases (EC Number 4) can break a bond or make a bond, but neither requires water
nor ATP.
Isomerases (EC Number 5) interconvert Isomers into each other. Ligases (EC Number 6) use ATP to make a bond.
is not is a Peroxidas Peroxidase. e. produce produc e H2O2 but it In Xanthine Oxidase Oxygenation means O2 is incorporated. Haemoglobin, there is Oxygenation. If Oxidation of Hb occurs, then metHb is formed. Mutase is Isomerase, not Transferase. All Synthases are Lyases. Exceptions : 1) Nitric Oxid Oxidee Synthase Synthase is an Oxidoreductas Oxidoreductasee 2) Glycogen Synthase and Citrate Synthase Transferase 3) ATP synthase Hydrolase
Oxygenases directly incorporate O2 into the substrate. All Hydroxylases are Mono-Oxygenases (Exceptions: Proline Hydroxylase and Lysyl Hydroxylase are Dioxygenases).
are known as as they incorporate one Mono-Oxygenases Mixed Oxidases [O] into the Substrate and other [O] into HFunction 2 to make H2O. Examples of Dioxygenases are Homogentisate Dioxygenase, Tryptophan Pyrrolase. Hydroxylase EC Number 1 Hydrolase EC Number 3 Hydratase EC Number 4 Phosphatase is Hydrolase K that [S] (substrate concentration) at which Velocity of reaction is half of V .
m
max
K m is Michaelis MentonConstant. Km isSignature of Enzymeandisinverselyproportional toAffinitybetweenEnzyme and Substrate. K chara cteristic of an enzym enzymee and an d its particular substrate, and reflects re flects the Affinity m is characteristic of Enzyme for for tha thatt Substrate. means a i.e. low concentration of substrate is needed to m Small High Affinity attainKhalf of Vmax.
34 : Enzymes Enzymes
Large Km means a Low Affinity i.e. high concentration of substrate is needed to attain half of Vmax. K m does not change with change in [E] or [S]. It is a Constant Value for each enzyme
substrate pair. Isoenzymes also have different K m values. Km is expressed in same units as [S] i.e. moles/L. Graph b/w Velocity and [E] Linear Graph. Graph b/w Velocity and [S] for Simple Enzymes Rectangular Hyperbola. Graph b/w Velocity and [S] for Allosteric enzymes/Regulatory enzymes Sigmoidal/S-shape . Graph b/w Velocity and Temp Bell shape. Graph b/w Velocity and pH Bell shape. Regulatory/Allosteric Enzymes are those which have extra Regulatory/Allosteric Site where regulator binds along with the Active Site. Allosteric Enzymes are usually Multi Subunit Enzymes. Allosteric Enzymes do not follow Michaelis Menton kinetics but follow Hill’s equation. Sigmoidal graph is because of a special property of Allosteric Enzymes i.e. . Cooperativity Cooperative Enzymes are more sensitive in their response to changes in Substrate Concentrations than other Enzymes. Most Rate Limiting Enzymes show Cooperative Kinetics. LineweaverBurkPlot or DoubleReciprocal Curveisamodification of MichaelisDoubleReciprocal Menton Graph (to convert Michaelis-Menton graph into a straight line graph). Enzymes get denatured at extremes of Temperature or pH. Hemoglobin Oxygen Dissociation Curve Sigmoidal/S-shape. Myoglobin Oxygen Dissociation Curve Rectangular Hyperbola. A Competitive Inhibitor binds with with the Free Free Enzyme Enzyme (at Active Active Site), reversibly reversibly,, to form Enzyme-Inhibitor Complex, that is catalytically inactive and cannot bind Substrate. Competitive Inhibitor K increase, V max same.
Oxamate is a Competitive inhibitor of Lactate Dehydrogenase. Physostigmine is a Competitive inhibitor of Cholinesterase . Sulphonamide (Antibiotic) competitivelyinhibits synthesis of Folic Acid in Bacteria. Methotrexate is a Competitive Inhibitor of Dihydro Folate Reductase. Dicumarol (Anticoagulant) is a Competitive Inhibitor of Vitamin K Analogue . A Non-Competitive Inhibitor binds at the regulato regulatory ry site and and induce induce a change in
the Active Site so that substrate cannot bind and thus decreases the velocity. Non-Com Non-Competitive petitive Inhibitor Inhibitor K and [S] same, V max decrease . Iodoacetate is a Non-Competitive Inhibitor of Glyceraldehyde-3-P D. Heavy metals and Dimercaprol are Non-Competitive Inhibitors of SH group. Di-Iso-Propyl Fluorophosphate is a Non-Competitive Inhibitor of Serine Protease. Uncompetitive Inhibitor Bot Both h K and Vmax decrease. Example of Uncompetitive Inhibitor is Acetylcholine which inhibits Placental ALP (Alkaline Phosphatase).
Enzymes : 35 35
Suicidal Inhibition is shown byspecial class of Inhibitors in whichenzyme converts
Inhibitor into a Reactive form in its Active Site. ExamplesofSuicidal/MechanismBasedInhibition: Allopurinolinhibiting Xanthine Oxidase and Aspirin inhibitingCycloxygenase . Feedback /En /End d Prod Product uct Inhibitio Inhibition n controls the amount of product the cell needs.
Functional Coagulation Plasma enzymes.Enzymes arethoseenzymes whichfunctioninplasmae.g. Blood
Non-Functional PlasmaEnzymes are Cell Derived Enzymes which functioninside
the cells or tissues e.g. ALT/SGPT. Cell damage damage leads to increased concentration of Non-Functional Plasma P lasma Enzymes Enzymes in plasma. Plasma = Physiologic Fluid. Serum = Prepared in lab. Serum = Plasma – ClottingFactors. ClottingFactors. Transaminases are used to diagnose Myocardial Infarction (SGOT) and Liver Diseases (SGPT). Alkaline phosphatase is used to diagnose Bone and Liver diseases and Hyperparathyroidism.
Prostate Cancer is diagnosed by Acid Phosphatase. Therapeutic Enzymes : Streptokinase, Asparaginase, Pepsin, α -1-antitrypsin, Uricase, Lactase, Trypsin and Chymotrypsin and Collagenase. Streptokinase/Urokinase catalyses the Lysis of Intravascular Clots e.g. in MI. Asparaginase/Glutaminase used in treatment of Acute Lymphoblastic Leukemia (ALL). Pepsin used in treatment of Chronic Ingestion and Pancreatic Insufficiency. α-1-Antitrypsin used in treatment of Emphysema that is caused by deficiency of α-1-antitrypsin. Uricase is used in treatment of Gout Gout. Lactase used in treatment of Lactose Intolerance and Penicillin Allergy Treatment. Trypsin and Chymotrypsin used for Pain & Inflammation. Collagenase used in treatment of Skin Ulcers Key Regulatory Reactions or Committed Steps of Metabolic Pathways are often subjected to Allosteric Regulation.
Allosteric Regulation of Enzyme When Modulator (M) bindss to the regul atory bind subunit, a conform conformation ation change is induced in the catalytic subunit, which enables the binding bindin g of Substrate (S) to the active site of enzyme
In the absence of Modulator (M), the substrate cannot binds to the catalytic subunit of enzyme
Most common Covalent Modification is Phosphorylation and Dephosphorylation.
36 : Enzymes Enzymes
Other Covalent Modifications
Enzyme Regulation by Covalent Modification
Methylation, Acetylation, Adenylation, ADP ADP Ribosylation, Uridylation. Phosphate group is added most commonly at the -OH group of Serine (less common commonat threonine).
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 Methylation
Enzymesactivein Phosphorylated stateare Glycogen Phosphorylase, Phosphorylase kinase, Key enzymes of Gluconeogenesis, HMG CoA Reductase Kinase and ATP Citrate Lyase. Enzymes active in Dephosphorylated state are Glycogen Synthase, Acetyl CoA Carboxylase, Pyruvate Dehydrogenase, HMG CoA Reductase and PFK-II. Compartmentalization of the cells gives a regulation to control theenzymatic activity. Enzyme synthesis can be induced or repressed at the level of genes. House Keeping or Constitutive Genes are those genes which are always active E.g. Enzymes of TCA cycle.
Inducible Genes are the genes which can be activated whenever required E.g.
Enzymes of Gluconeogenesis.
Ubiquitin Proteosome Path Pathway way (UPP) is ATP ATP Dependent Pathwayfor degradation
of proteins/ enzymes. UPP occurs occurs in Cytoplasm and Nucleus and is for damaged or short lived proteins. prote ins. Ubiquitin prote protein in is highly highly conserved, conserved, small, small, globular, globular, non non enzyme enzyme protein protein and is recycled. ε-amino of Lysine in protein is attached to C-teminal Glycine of Ubiquitin. ATP Independent Proteolytic Pathway occurs in Lysosomes and Enzyme used is Hydrolase. Proteins having Amino Acids such as Arginine and Acetylated Alanine at N-terminal ends have short half lives and are rapidly degraded. Proteins rich in Proline, Glutamate, Serine And Threonine ( PESTsequence ) are also degraded.
Zymogens are inactive precursors ofenzymes.
CHAPTER
5
ACIDS AMINO ACIDS
AMINO ACIDS – CLASSIFICATION CLASSIFICATION & METABOLISM Amino group (-NH2) is always on left side and Acid group (-COOH) is always on right side in an Amino Acid. Any compound having Asymmetric Carbon shows both Optical and Structural Isomerism. All Amino Acids have 1 Asymmetric Carbon except - Glycine, Threonine and Isoleucine. Glycine has no asymmetric carbon.
Threonine & Isoleucine have 2 asymmetric carbons. CH3
OH
NH2
CH
C
CH3 NH2 COOH
CH3 CH2
CH
COOH
C
H
H
threonine
isoleucine
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. pI is Isoelectric pH that pH at which Zwitter ions exist. At pI (Isoelectric pH), a protein will precipitate.
basic group group
acidic group
H
H2N
C
COOH COOH
R
amino acid
H
+H N N 3
C
COO
R
zwitterion
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. Amino Acid abundant in Protein L-form. Amino Acid abundant in Body L-form.
Amino Acid present in Protein always ‘L’ form and always α. Amino Acid present in Body Both ‘L’ and ‘D’ forms.
38 : Amino Acids Acids
Free Amino Acids may be L-form or D-form. Free Amino Acids may beα/β/Y. Essential Amino Acids are those that cannot be synthesized in our body, hence are
required in diet.
Non-Essential Amino Acids are those that can be synthesized in our body, hence
are not requiredAmino in diet. Acids are those that can be synthesized in our body but to Semi-Essential
some extent. A total of 20 Amino Acids are required in body for synthesizing Proteins of which 8 are Essential. st nd 21 and22 Amino AcidareSelenocysteine(givenbycodon UGA) and Pyrrolysine ( given by codon UAG) respectively. These two Amino Acids are formed by modification of Stop Codons which which is a Co-Translational Co-Transla tional Modif Modificatio ication. n. 22 Amino Acids are encoded by DNA i.e. they are not formed by Post Translational Modifications. Pyrrolysine NH2
O
OH
X N H O
N
Lysine
NH2 OH
H2 N O
Precursor Amino Acid for Pyrrolysine is Lysine. Pyrrolysine is not found in humans. Precursor Amino Acid for Selenocysteine is Serine.
H
H2N
SeH
SH
OH
O
OH Serine Seri ne (Ser)
H
H2N
O
OH Cysteine (Cys)
H
H2N
O
OH Selenocysteine (Sec)
Aromatic Amino Acid with basic proper properties ties is Histidine. Aromatic Amino Acid with – OH group is Tyrosine. OH 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 Acids : 39 Arginine
Histidine
O H2 N
CH
C
O OH
H2N
CH 2
CH
C
OH
CH 2
CH 2 N CH 2 NH NH C
NH
NH2
Histidine can be formed in Adults but not in in Children. Children. Amino Acid responsible for Flexibility of Proteins is Glycine (because of its smallest side chain).
is not found Glycine in β-turns/bends . in α -helix, but found Glycine is the simplest, smallest, Non-
Essential, Non-Polar, and Glucogenic Amino Acid.
H H2N
C COOH H glycine
Glycine can be synthesized from:
1) CO2 and NH4+ by Glycogen Glycogen Synthase Synthase (N5, N10 Methylene THF involved) 2) Fro From m Glyoxylate by Glycine Amino Transferase/ Glycine Glycine Transaminase (PLP required). 3) Fro From m Serine by b y Serine Hydroxy Hydroxyll Methyl Methyl Transferase Trans ferase (reversible reaction) rea ction) – PLP PLP 5 10 and Folic Acid required (N , N Methylene THFinvolved). 4) Fro From m Threonine Thre onine by Threonine Aldo Aldolase. lase.
Glycine Cleavage System occurs in Mitochondria and is a Major Pathway for Glycine Degradation. Primary Hyperoxaluria is defect in Glycine Glycine Transaminase associated a ssociated with Impaired Glyoxylate to Formate conversion, which diverts excess of Glyoxylate to form Oxalate which can form Oxalate Stone in Kidneys. Oxalate in urine can appear in B6 deficiency.
Alanine (Non-Polar) is the most Glucogenic
Amino Acid (3C).
CH3 H2N C COOH H Alanine
All branched chain Amino Acids are Essential and Non-Polar.
Isoleucine is the most Non Non-Polar Polar Amino Amino Acid Acid and is both both Glucogenic Glucogenic and Ketogenic. Ketogenic.
40 : Amino Acids Acids Amino Acid that can act as a fuel for Brain is Isoleucine. Leucine is purely Ketogenic. Valine is Glucogenic.
-
-
+
C H CH
H3C CH3
Valine
COO +
+
H 3N
-
COO
COO H 3N N
C H CH
N C H 3N H C
H CH3
CH CH
CH2
H3C CH3
CH3
Leucine
Isoleucine
Maple Syrup Urine Disease (MSUD) is Autosomal Recessive disease in i n which there is a defect in the Oxidative Decarboxyl Decarboxylation ation step (in the catabolism of Branched Chain Amino Acids).
Enzyme defect in MSUDis branched chain α-Ketoacid Dehydrogenase/ Branched Chain α-Keto Acid Decarboxylase (Multi enzymecomplex).
Clinicalfeatures Clini calfeatures of MSUD are Burnt Sugarlike Odour Of Urine, Ketosis, Vomiting,
Feeding Problems, Mental Retardation, Abnormal Muscle Tone If MSUD is not treated , then Coma or Death may occur. Patients with rare Thiamine Dependent Variant of MSUD give response when large doses of Vitamin B1 isgiven. Isovaleric Acidemia – Defect in FAD Dependent Dehydrogenase (Isovaleryl CoA Defect Dehydrogenase) which specificallydegrades Isovaleric Acid (involved in Metabolism of Leucine) – Sweet feet odour of urine.
All AliphaticAminoAcids (Valine, Isoleucine, Leucine, Alanine, Glycine) are NonPolar.
Least Non Non-Polar Polar is Glycine.
Glycine Metabolism is linked with THF & Vitamin B6. Primary Hyperoxaluria is defect in enzyme Glycine Transaminase. Caus Causes es of SecondaryHyperoxaluria are Vitamin B6 deficiency, deficiency, Vitamin Vit amin CToxicity CToxici ty & Ethylene Eth ylene Glycol Glycol Poisoning.
In Non-Ketotic Hyperglycinemia (or Glycine Encephalopathy), there is defect in Glycine Cleavage System.
reabsorption on of Glycine due to defective transporter InGlycinuria , there is defective reabsorpti for Glycine & Proline.
Uses of Glycine Formation of Haem, Glutathione, Serine, Creatine, Creatinine,
Choline, Betaine and Conjugation of Primary Bile Acids.
Creatine is formed from Arginine & Glycine and SAM acts as a Methyl Donor. Choline and Betaine Metabolism is interrelated with Folate metabolism.
Amino Acids 41 Acids : 41
Aromatic Amino Acids: Phenylalanine, Tyrosine, Tryptophan.
Phenylalanine is converted to Tyrosine Tyrosine by enzyme enzyme Phenylalanine Hydroxylase (mono-oxygenase), Tetrahydrobiopterin (THB) & NADPH is requiredas requireda s Coenzym Coenzyme. e. Catecholamines are Dopamine, Epinephrine &Nor-Epinephrine. First Catecholamine to be synthesized is Dopamine.
Catecholamine with Methyl Group is Epinephrine.
Synthesis of catecholamines (Dopamine, NE and E) from tyrosine
Epinephrine formation occurs in Periphery (not in CNS), 80 % of it occurs in Adrenal Medulla. Catabolic end product of Epinephrine & Nor- Epinephrine is VMA (Vanillyl Mandelic Acid). Rate Limiting Enzyme in Catecholamine Synthesis is Tyrosine Hydroxylase Precursor of all three Catecholamines are L-DOPA (Di-Hydroxy Phenyl Alanine). Catabolic End Product of Dopamine is Homovanillic acid.
42 : Amino Acids Acids Normal VMA levels Normal levels are 2-6 mg/ 24 hours. VMA levels are increased in Pheochromocytoma & Neuroblastoma of Adrenal Glands. THB also required for Tyrosine to DOPA conversion, Tryptophan to 5-OH Tryptophan conversion and Arginine to Citrulline conversion.
THB
is required mainly for . Phenylalanine, Tryptophan, Hydroxylases Tyrosine are both Glucogenic and Ketogenic. Phenylketonuria (PKU) is due to defect in enzyme Phenylalanine Hydroxylase. Phenylketonuria patient is normal at birth.
Biochemistry of Phenylketonuria
Clinical features of Phenylketonuria are Mousy/Musty odo odour ur (due to Phenylacetate), Severe Mental Ment al Retardation, Retarda tion, microcephaly microcephaly,, exaggerated tendon reflex, seizures, hyperactivity, rash, wide spaced spaced teeth, Hypopigmentation (Melanin not formed because of Tyrosine
deficiency)- fair skin, blueeyes. Screening of Phenylketonuria is done by Tandem Mass Spectrometry (accurate, precise, low false false positiveresu positiveresults). lts).
Phenyl ketonuria Mousy odor Hypopigmentation Seizures
Severe mental retardation
Gutherie’s Test (Bacterial Inhibition Assay) for Phenylketonuria is now replaced by
Tandem Mass Spectrometry.
Hyperphenylalaninemia can also occur from THB deficiency. Tyrosine is used for the synthesis of Catecholamines, Thyroid Hormone and the
pigment Melanin. Melanin.
Amino Acids 43 Acids : 43
Albinism (milky white skin, white hair, red eye color) color) is absenc absencee of pigment
Melanin from skin, hair and eyes. Enzyme deficient is Tyrosinase (an Oxidase and Cu required). Oculo Cutaneous Albinism associated with defects in Vision and Photophobia. In Vitiligo, enzyme Tyrosinase is normal but there is lack of Melanoblasts in regional areas. Alkaptonuria is associated with defects in Catabolism of Tyrosine and
Phenylalanine. Alkaptonuria (Autosomal Recessive) is part of Garrod’s Tetrad. Benedict’s Test is positive in Alkaptonuria due to Homogentisic Acid which is Reducing in nature. Fresh urine of Alkaptonuria patients is normal color. But urine on long standing turns black due to oxidation of Homogentisic Acid. There is no Mental Retardation in Alkaptonuria. Polymerization of Homogentisic Acid forms forms Alkapton Bodies which gets accumulated accum ulated in i n Cartilages giving Bluish Black Connective Tissues. Enzyme deficient in Alkaptonuria is Homogentisate Dioxygenase.
Characteristics of Alkaptonuria are Dark staining of Diapers, Deposition of Black
Pigments in joints, Cartilage and Collagenous Tissue(Ochronosis) Nitisinone is used in treatment of Alkaptonuria. Nitisinone inhibits PHPP (Para Hydroxy Phenyl Pyruvate) Hydroxylase. Glutathione is required in the Catabolism of Phenylalanine & Tyrosine. Homogentisic Acid is an intermediateinthe Catabolism of Tyrosine. – defective Tyrosinemia Type I or Tyrosinosis is most common Tyrosinemia Fumaryl Aceto Acetate Hydrolase – Terminal Terminal Enzyme Enzyme iin n Tyrosine Catabolism – characterized by Boiled Cabbage/Rancid Butter like Odour of Urine, also known as Hereditary Tyro Tyrosinem sinemia ia or Hepato-RenalTyrosinemia. Tyrosinemia Type II is known as Richner Hanhart Syndrome or Oculo-Cutaneous Tyrosinemia is deficiency deficien cy of Tyrosine Transaminase.
Tyrosinemia
isNeonatalTyrosinemia -deficiencyof PHPP TypeIII PHPP(ParaHydroxy Phenyl Pyruvate) Hydroxylase. Tryptophan Pyrrolase is a Dioxygenase and contains haem. Uses of Tryptophan – Formation of Serotonin, Melatonin & Niacin. Formation Rate Limiting Enzyme in Niacin synthesis is QPRTase (Quinolate Phospho Ribosyl Transferase). 60 mg Tryptophan forms 1 mg Niacin. Serotonin is synthesized in Intestine & Placenta and is a Neurotransmitter responsible for Mood Elevation and Temperature Regulation. Melatonin is Acetyl Methyl Serotonin. Melatonin is synthesized in Pineal Glands and is the Neurotransmitter responsible for Circadian Rhythm.
Excretory
product of Serotonin is 5-HIAA (Hydroxyl Indole Acetic Acid). Tryptophan forms 5-OH Tryptophan which is converted to Serotonin (5-OH Tryptamine) and then Melatonin.
Acids 44 : Amino Acids Tryptophan also forms Niacin/ Vitamin B3 (known as Atypical vitamin). Atypical Vitamins are Vitamin B3 & Vitamin D. Vitamin B-6/ PLP deficiency leads to increased Xanthurenic Acid. 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.
Massive Aminoaciduria (mainly Tryptophan) without a corresponding increase in
Plasma Amino Acid level is characteristic of Hartnup’s Disease. Mutation of Hartnup’s Dis ease is on gene SLC6A 19. In Carcinoid Syndrome, Tryptophan used us ed to form excess Serotonin, so Tryptophan to Niacin conversion not occurring, so Pellagra occurs. Catabolic end product of Histidine is Glutamate formed via FIGLU. Histidine has maximum Buffering Capacity because Histid Histidine ine is the the only Amino Acid which can ionize within the physiological pH range.
Arginine > Lysine > Histidine (in terms of Polarity). All Basic Amino Acids (Histidine, Arginine, Lysine) and Acidic Amino Acids( Aspartic Acid, Glutamic Acid) are Polar.
Arginine is Glucogenic (Catabolic End Product – α-Ketoglutarate ).
Asparagine synthesis require ATP and Glutamine is – NH2 donor. All Acidic Amino Acids and their Amides are Glucogenic.
COO
COO +
H3N
+
C
H
H3N
CH
CH2
CH2
COO COO
CH CH 2
COO
COO
+
+
H3N C H
H3N
CH2
CH2
C
CH2
H2N
O
C
COO COO H2N Aspartate [D] (Asp)
Glutamate [E] (Glu)
CH
Asparagine [N] (Asn)
O
Glutamine [Q [Q]] (Gln)
Asparagine is the site for N-glycosidic bonds. Serine and Threonine are – OH containing Amino Acids. Also the site for OH
O-glycosidic bonds. NH2 HO
CH2
CH Serine
COOH
CH3
OH
NH2
CH
CH
Threonine
COOH
Amino Acids 45 Acids : 45 Cysteine and Methionine are Sulphur containing Amino Acids.
Cysteine
Methionine
Cysteine is oxidised to Cystine (Disulphide bond formation). Methionine forms SAM (S-Adenosyl Methionine)- Methyl donor.
MAT (Methionine Adenosyl Transferase) converts Methionine to SAM (S-Adenosyl
Methionine). MAT 1 & 3 is in Liver. MAT-2 is in Extra-Hepatic Tissues. buttheydon’thave don’thave Phosphate. Phosphate. SAMand Coenzyme Aare High EnergyCompounds butthey Cysteine is not an Essential Amino Acid as long as Methionine is available in diet. Tyrosine is not an Essential Amino Acid as long as Phenylalanine is available in diet. Cystine is made up of two molecules of Cysteine. SH group but Cystine SH bond. Cysteine has – Cystine has disulfide bond.
Acids 46 : Amino Acids
Homocystine is made up of two molecules of Homocysteine. Homocysteine is increased in Blood in vitamin B6, B9 or B12 deficiency. In urine,
Homocystine is increased. In Type I Homocystinuria, Cysteine becomes Essential. In Type II Homocystinuria, Cysteine can be synthesized in body.
For
the formation of Cysteine, two Amino Acids are required i.e. Methionine & Serine and one vitamin required is Vitamin B6. Cyanide Nitroprusside Test is positive for Homocystinuria, Cystinuria & Cystinosis. In Cystinuria , there is a defect in Dibasic Amino Acid Transporter. In Cystinosis, there is defect in Cystine Transporter (Cystinosin) in Lysosomes, so it is a generalized Lysosomal Storage Disorder. Cystinuria Most common genetic errorin Amino Acid transport - ‘COAL’ carrier defective - 4 Amino Acids: Cysteine, Ornithine, Arginine, Lysineappearinurine.
All Amino Acids have h ave Primary Amino Group except Proline having Secondary Secondar y Amino Amino Group. Proline (Imino group) can never b bee accommodated in α-helix. Catabolic end product of Proline is Glutamate. So Glucogenic:
Proline
Wheat lacks Lysine Pulses lack Methionine
AMINO ACID CATABOLISM & UREA CYCLE
Transamination is the transfer of NH2 group from Amino Acid to corresponding
α-Keto acid. Transamination reactions are reversible and require PLP (derivative of Vitamin B6) as coenzyme. Removal of Amino Group from Amino Acids form α-Keto acids. Transamination is the first reaction in the Catabolism of Amino Acids. On addition of – SGOT/SGPT and NH2 group, α-Ketoglutarate forms Glutamate by SGOT/SGPT further forms Glutamine by enzyme Glutamine Synthetase (requires ATP). On addition of – NH2 group, Oxaloacetate forms Aspartate and further forms Asparagine. Glutamate is regarded as collector of Amino Groups. Glutamate is the only Amino Acid that can undergo Oxidative Deamination. Transdeamination is coupling of Transamination (occurs in i n all organs) & Oxidative Deamination (occurs in liver). 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. 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 Acids : 47
SGPT: Serum Glutamate Pyruvate Transaminase – also also known as ALT (Alanine Transaminase )- Alanine loses amino group and forms Pyruvate and α-Keto
Glutarate takes ta kes amino group and forms forms Glutamate. Glutamine Synthetase converts Glutamate to Glutamine (ATP used) and Glutaminase converts Glutamine to Glutamate (no ATP used).
Glutamine is a Non-Toxic Storage and Transport Form of Ammonia in Blood. 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 NA D or NADP. NADP. Glutamate forms GABA (Gamma Amino ButyricAcid). Actiavtors of GDH : NAD, ADP & GDP. Inhibitors of GDH : NADH, ATP & GTP. Major transporter of NH3 from Body & Brain is Glutamine and from Muscles it is
Alanine. Amino Acids which cannot form Glutamate and cannot undergo transamination are Proline, Hydroxy- Proline, Lysine and Threonine. Transport of ammonia from Transport Glucose– Alanine cycleisalsoknownas Cahill cycle –
Muscles to Liver – occurs occurs in Fasting Muscles.
Excess Ammonia increases Glutamineand decreases α-Ketoglutarate and Glutamate
and BUN (Blood Urea Nitrogen) is also decreased. Clini Clinical cal features of Hyperam Hyperammonemia monemia are Lethargy, Mental Confusion, Agitation, Blurred Vision, Slurred Speech, Cerebral Edema, Vomiting, Fine Tremors, Hyperventilation and if not treated then. Ammonia Encephalopathy – Increased Glutamine is osmoticallyactive andleadsto Increased cell swelling, specially Brain cells – Symptoms Symptoms are Cerebral Oedema and Vomiting.
Acids 48 : Amino Acids
Urea Cycle occurs in Liver. Urea Cycle occurs in both Mitochondria and Cytoplasm.
RateLimiting Enzyme of Urea Cycle – Carbomoyl Carbomoyl Phosphate Synthetase–I (CPS-I). Arginine is the immediate precursor ofUrea. Urea is synthesized by Arginase. Aspartate is transported from Mitochondria to Cytoplasm by Citrin Transporter. Ornithine and Citrulline Antiport is present pres ent in IMM, IMM, which tra transport nsportss
Citrulline from Mitochondria to Cytoplasm Cytoplasm and Ornithine is transported into the Mitochondrial Matrix. th NAG Synthase is regarded as 6 enzyme of Urea Cycle, which synthesize NAG (NAcetyl glutamate). NAG is the Allosteric Activator of CPS-I (Rate Limiting enzyme of Urea Cycle). NAG Synthase is activated by Glutamate & Arginine Arginine andit an dit is inhibited by N-Acetyl
glutamate. 4 ATPs are used to synthesize one molecule of Urea. Ornithine has a catalytic role in Urea Cycle. Carbon of urea is derived from CO 2 or bicarbonate (HCO -), Two 3 nitrogens are derived from aspartate and ammonia. Urea Cycle Enzyme deficient in Brain: OTC (Ornithine Trans Carbamoyalase ). Urea Cycle Enzyme deficient in Kidneys : Arginase. End product of Urea Cycle in Kidneys: Arginine. Major source of Arginine (a Semi-Essential Amino Acid) is Kidneys. Main Clinical Features of Urea Cycle Disorders are Hyperammonemia, Encephalopathy And Respiratory Alkalosis. OTC (Ornithine Transcarbamylase) Deficiency (Orotic Acid, OMP, UMP increased) Type-II e-II. isthemostcommon Urea Cycle Cycle Disorder. It isknown asHyperammonemia Typ Hyperammonemia Type I is deficiency of CPS-I.
Amino Acids Acids : 49 49 Hyperammonemia Type I & Hyperammonemia Type II are most severe Urea Cycle defects as ammonia is present in inorganic form which is highly toxic. contraindicated d Arginine is the first line treatment of Urea Cycle Disorders but it is contraindicate in Hyperargininemia. Sodium Benzoate and Phenylbutyrate are Ammonia Scavenging Agents.
Deficiency of enzyme Arginosuccina Arginosuccinate te synthetase leads to Citrullinemi Citrullinemiaa Type I. Defect in Citrin Transporter (transports aspartate) leads to Citrullinemia Type II. Phenyl Butyrate is more effective than Sodium Benzoate in treating Urea Cycle disorders. Increased Gluconeogenesis from Amino Acids increase Ureaformation.
PROTEINS – BONDS BONDS & STRUCTURES
Proteins are polymers of Amino Acids
Amino Acids are joined by strong Covalent
Amide/ Peptide bonds. If Amide bond is present in proteins then it is
bond. d. called Peptide bon
Protein foods
Peptide bond rigid and planar partial double bond character character “trans” configuration.
Unsaturated Fatty Acid Double Bond “cis”. If < 50 Amino Acids in a chain known as Peptide. If > 50 Amino Acids in a chain, then known as Polypeptide or Protein. Protein digestion begins in Stomach. HCl from parietal cells just denature the Proteins and activates Pepsinogen toPepsin. Pepsin is an Endopeptidase. Pancreatic Enzymes are Trypsin, Chymotrypsin, Elastase and Carboxypeptidases A+ B. Endopeptidases cut at the Carboxy Terminal of Specific Amino Acids. Exopeptidases cut at the C- terminal of Protein. Carboxypeptidases A: Preferablyacts on Alanineand Branched Chain Amino Acids (BCAA). Carboxypeptidases B: Preferably acts on Basic Amino Acids e.g. Arginine, Lysine. Small Intestinal Enzymes are Aminopeptidases, Dipeptidases and Tripeptidases
(Also secreted as Zymogens). Aminopeptidases: An Exopeptidase, cut peptide bond at N-terminal of Protein.
Acids 50 : Amino Acids
Dipeptidases and Tripeptidases: Cleaves Di and Tri peptides into their individual
Amino Acids, which are absorbed.
Primary Structure: Sequence of Amino Acids joined by Peptide bonds. Secondary structure: Folding of Primary Structure e.g. α -helix, β-sheet, β-turn/
bends, α-chain in Collagen.
Each turn of α-helix contains 3.6 Amino Acids. α-helix is Rigid and Right handed - Only Interchain Hydrogen bonds present. β-sheet has both Interchain and Intrachain Hydrogen bonds – fully extended chain fully – may be parallel or anti- parallel.
Tertiary Structure: Single fully folded Functional Polypeptide chain. Quaternary Structure: More than one fully folded Functional Polypeptide chains.
Monomeric proteins do not contain Quaternary structure.
Proline is never found in α-helix. Proline and Glycine are found in beta turns.
Amino Acid with Bulky R groups : Tryptophan.
Amino Acids Acids : 51 51 Amino Acid with Branch Chain at beta carbon : Valine. Mass Spectroscopy is the best technique to determine the Primary Structure of Proteins.
X-ray crystallogra crystallography phy is the best technique to detect Secondary, Tertiary or
Quaternary Structure of Proteins.
NMR Spectrometry is thebesttechniquetodetect Secondary, Tertiaryor Quaternary
Structure of Non-Crystallizable Proteins or Membrane Proteins
X-ray crystallogra crystallography phy detects 3-D Structure of any Macromolecule.
Most powerful technique for screening inborn errors of metabolism is Tandem
Mass Spectrometry. 2 Tandem Mass Spectrometry, also known as MS/MS or MS in the first stage of MS, precursor precurs or (characteristic of given anal analyte) yte) is selected, then fra fragmen gmentatio tation n occurs occurs by collision collision induced dissociatio dissociation n to produce ions. ions. Then in second stage stage of MS, ions are separated according to mass to charge ratio, and then detected. The fragments then reveal re veal aspects of the chemica chemicall structure struct ure of the precursor pre cursor ion. Denaturation of Proteins is sometimes reversible. Denaturing Agents are Heat, Urea, Guanidine, UV rays, IR rays, Strong Acids or Alkalis. Coagulation is clumping of Denatured Protein and is always irreversible.
CHAPTER LIPIDS LIPIDS
6
LIPID BIOCHEMISTRY
Fatty Acid consists of a Hydrophobic Hydrocarbon Chain (Non-Polar) and a Hydrophilic Carboxyl Group(Polar) whichisionized 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.
Terminal Methyl Group in a Fatty Acid is called ω-carbon (omega).
C2 in case of Fatty Acid is called α-carbon (alpha).
C3 in case of Fatty Acid is called β-carbon (beta). C4 in case of Fatty Acid is called -carbon (gamma). Acid + Alcohol = Esterbond. Fatty Acid (Polar) + Alcohol (Polar) = Fat (Non-polar). Acid + Amino = Amidebond. Simple Lipids are only made up of 2 components i.e. Fatty Acid (FA) and Alcohol. Complex Lipids are made up of three components Fatty Acid, Alcohol and Other Component (Phosphate or Carbohydrate) e.g. Phospholipids & Glycolipids. Alcohol in Simple Lipids is Glycerol (3C). Mono-Acyl Glycerol (MAG) : One Fatty Acid + Glycerol. Di- Acyl Glycerol (DAG) : Two Fatty Acids +Glycerol. Tri- Acyl Glycerol/Neutral Fat/Triglycerides (TGs) : Three FAs + Glycerol Mono Acyl Glycerol & Di Acyl Glycerol is Amphipathic . But Tri Acyl Glycerol is Non Polar. Polar. TGs are the main lipids presen presentt in diet diet and also the main Storage Form of Lipids in the body (in Adipose Tissues). Hormone Sensitive Lipase presen presentin tin Adipose Adipose Tissues Tissues breaks down down TGs. TGs. It is ac acti tive ve in Fasting state.
Phospholipids/ Phosphoglycerides = Alcohol + Fatty Acid + Phosphate. Phospholipids are of two types: GlyceroPhospholipids and Sphingo-Phospholipids. Parent alcohol is Glycerol (3 C) in Glycero-
Phospholipids.
Lipids 54 : Lipids Parent alcohol is Sphingosine (20 C) in Sphingo-Phospholipids. Phosphatic Acid = Glycerol +2 Fatty Acids +Phosphate. Phosphatidi Phosphatidicc Acid is the simplest Phospholipid and is also the precursor of all Phospholipids. Lecithin is Phosphatidyl Choline (Glycerol + 2 Fatty Acids + Phosphate + Choline).
Most abundant Phospholipid in HDL isLecithin. Cephalin is Phosphatidyl Ethanolamine (Glycerol + 2 Fatty Acids + Phosphate + Ethanolamine). Lecithin and Cephalin are the most abundant Phospholipids in most eukaryotic cells. Phosphatidyl Serine is involved in Apoptosis. SAM (S-Adenosyl Methionine) is used in the synthesis of Lecithin from Cephalin. Lecithin is the largest body store of Choline and most abundant Phospholipid of Cell Membrane. L/S ratio (Lecithin : Sphingomyelin ratio) > 2 indicate Fetal Lung Maturity, which occurs at 32 weeks ofgestation. Dipalmitoyl Lecithin is used as a Surfactant in Lungs. Cephalin is present Brain/CNS.
Phospholipases arethe enzymes which break down Phospholipids. These are of four types. Phospholipase A1 breaksthe first fir st FA i.e i.e.. from C1. Phospholipase A2 breaks the FA from C2. Phospholipase C breaks Phosphory Phosphoryll base fromrest fromrest of the Molecule. Molecule. Phospholipase D acts between Phosphate and the base. Cardiolipin/ Phosphatidyl Glycerol = Glycerol + 2 Phosphatidic Acids. A Phospholipid which can be Antigenic is Cardiolipin (present in Inner Mitochondrial Membrane). Barth Syndrome – a rare X-linked Disorder, occurs due to defect in Cardiolipin Modelling. Patients have Muscle Weakness, Neutropenia And Cardiomyopathy. 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). Sphingosine is a 20-C Amino Alcohol, which is synthesized from Serine and Palmitate. Ceramide = Sphingosine + FA (mostly Long Chain FA). Ester bond present usually in all Fats, but Ceramide is a lipid having Amide bond. Sphingomyelin = Sphingosine + FA + Phosphate + Choline. For Sugar Activation, UDP (Uridine Diphosphate) is used in Carbohydrate Synthesis. Similarly, in Phospholipid Synthesis, CDP (Cytidine Diphosphate is used). Glycolipids or Glycosphingolipids are the source of ABO Blood Group Antigens, where Carbohydrate is the Antigenic Determinant. Glycolipid/Cerebrosides = Alcohol + Fatty Acid + Carbohydrate (Glucose/
InGalactose). Glycolipids, always Sphingosine Alcohol is present (not Glycerol). Hence also known as Glycosphingolipids .
55 Lipids : 55 If Glucose is added to Ceramide, then it is known as Gluco-cerebroside or GlucosylCeramide. If Galactose is added to Ceramide, then it is known as Galacto-cerebroside or Galactosyl-Ceramide. Gluco-cerebrosides are never found in CNS but Galacto-cerebrosides are always
found in CNS. If Oligosaccharide Chain containing Sialic Acid/N-Acetyl Neuraminic Acid (Polar Component) is further added to Cerebrosides, then known as Gangliosides (Complex Glycolipids). Gangliosides are found in Ganglion Cells of CNS and are never found in Liver. Simplest Ganglioside is GM3 Ganglioside. GM1 acts as receptor for Cholera Toxin in Intestine. IfSphingomyelinaccumulates,thenitis Neimann’sPickdisease . Enzymedeficiency is Sphingomyelinase whic which h is a kind of Phospholipase C. If Glucocerebrosides accumulates then it is Gaucher’s Disease. Enzy Enzyme me deficien defi ciency cy is Glucocerebrosidase. There is no Mental Retardation in Gaucher’s Disease. Gaucher’s disease is the most common Lysosomal Storage Disease.
β-Galactosyl Ceramidase deficiency leads to Krabbe’s disease. Fabry’s disease (α-Galactosyl Ceramidase deficiency) is XR (X-Linked recessive). If Gangliosides accumulate then it is Tay Sach’s Disease . Enzyme deficiency is Hexosaminidase A. There is no Hepatosplenomegaly in Tay Sach’s disease. Sphingolipidoses are Niemann’ Niemann’s 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. Sphingolipidoses with no Mental Retardation is Gaucher’s Disease, Fabry’s Disease. Sphingolipidoseswith nocherry red spots is Gaucher’s Disease, Fabry’s Disease. VLCFA (Very Long Chain Fatty Acids) (> 22C) are usually present in Brain. Coconut and Coconut Oil has highest content of Medium Chain Fatty Acids. Olive Oil has Long Chain (C16-18) Unsaturated FA. Butter has Long Chain (C16-18) SaturatedFA. Beef Fat has Long Chain ( > C18) Saturated FA. Number Number of Hydrogens 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 Hydrogens.. Usually unsaturated FAs have Double Bonds in ‘cis’ configuration but on prolonged heating, it gets converted to ‘trans’ configuration. 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. Monounsaturated Fatty Acids (MUFAs): one Double bond present e.g. Oleic acid (18C, one double bond). Polyunsaturated Fatty Acids (PUFAs) are known as Essential FAs as they are essential in diet. PUFA PUFAs are divided into ω-3 (omega 3) and ω-6 (om (omega ega 6) categories. cat egories.
ω-3 Essential FAs are Cervonic Acid, α -Linolenic (18 C, 3 Double bonds) acid and
Timnodonic Acid (20 C, 5 Double bonds).
Lipids 56 : Lipids
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). Most essential Fatty Acid is Linoleic Acid. α-Linolenic Acid is the precursor of ω-3 category. Linoleic Acid is the precursor of ω-6 category.
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. 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. 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. Humans cannot introduce double bonds beyong Δ9 (delta 9) position. Antioxidants are added in oils to prevent Rancidity (Hydrolytic cleavage of double bond leading to formation formation of of Short Chain Chain Fatty Fatty Acids Acids & Aldehydes). Aldehydes).
LIPOPROTEINS Lipids present in Lipoproteins are TGs, Phospholipids, Free Cholesterol (unesterified) and Cholesterol Ester. TGs and Cholesterol Ester are neutral or totally Non-Polar NonPolar and lies in the core. Choleste Cholesterol rol and PLs PLs are Amphipathic Lipids and lie towards the periphery. periphery. 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. 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. Density is directly proport proportionalto ionalto Percentage of Proteins andinverselyproportional to TG Content and Size. HDL has maximum density, so smallest size, has minimum TG content and maximum Proteins. In circulation, HDL donates Apo C and Apo E to Chylomicron and VLDL so that they are converted to Chylomicron remnant and VLDL remnant. Chylomicrons are largest insize. Lipoprotein Lipase enzyme is synthesizedby Adipose Tissues, Cardiacand Skeletal Muscles. This enzyme is activated byInsulin. Chylomicrons carry Exogenous Lipid (or Exogenous TGs) from Intestine to Peripheral Tissues. Chylomicron remnant is degraded in Liver.
Lipids : 57
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. Cholesterol is transported to Peripheral Tissues by LDL. HDL takes Cholesterol from Peripheryto Liver: also known as Reverse Cholesterol
Transport. 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. Main Ligand for LDL-Receptor is Apo B-100 and minor Ligand is Apo E. Ligand for HDL-Receptor is ApoA-I. Ligand for Chylomicron remnants & VLDL remnants is Apo E. Apo C-I and Apo C-II activate Lipoprotein Lipase. Apo C-III inhibits Lipoprotein Lipase. Apo A-I activate LCAT. Apo A-II inhibits LCAT.
HDL (cardioprotective) is known as α-Lipoprotein or Lipoprotein -A. Deficiency of HDL leads to Tangier’s Disease. LDL is known as β-Lipoprotein and is increased in DiabetesMellitus. VLDLisknownasPre- β-Lipoprotein. IDLisknownas Broadβ-Lipoprotein. 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 for Cardiovascular diseases. It contains LDL and Apo ‘a’. Lipoprotein A or HDL preventAtherosclerosis. Lipopro Lipoprotein tein ‘a’ causes Atherosclerosis (as it contains LDL). Insulin enhances the action of Lipoprotein Lipase but only in the capillary beds of
Adipose tissue.
Hormone Sensitive lipase is activated by Glucocorticoids, Glucagon, Epinephrine,
Nor-Epinephrine,, Dopamine Nor-Epinephrine Dopamine and Growth Growth Hormones. Hormones. HDL has maximum Electrophoretic Mobility. Chylomicron has Apo B-48.
Lipoprotein Lipoprotein
Lipid Lipid
Protein 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 HDL
Cholesterol Cholesterol
Apo B-100 + Apo E Apo-A, Apo-C and Apo-E
58 : Lipids Lipids Chylomicron remnant has Apo B-48 + Apo E. VLDL has Apo B-100. VLDL remnant/IDL and LDL has Apo B-100 + Apo E. FamilialHyperchylomicronemia or TypeI Hyperlipoproteinemia is characterised by increase in Chylom Chylomicro icrons ns ( ) and VLDL ( ) and TGs ( ) while Cholesterol
is normal.
Familial Hyperch Hypercholesterole olesterolemia mia or Type II-a Hyperlipoproteinemia is characterised
by increase in LDL ( ) and Cholesterol () while TGs are normal. Familial Hyperlipoproteinemia or Type II-b Hyperlipoproteinemia is characterised by increase in VLDL () , LDL ( ), Cholesterol () and TGs ( ). Dysbeta-Lipoproteinemia or Type III Hyperlipoproteinemia or Broad-Beta Disease is characterised charact erised by b y increase in Chylomic Chylomicron ron remnant ( ) , V LDL remnant remn ant (), Cholesterol ( ) and TGs T Gs (). In Type I Hyperlipoproteinemia, there th ere is a defect in Lipoprotein lipase or Apo C-II. In Type II-a Hyperlipoproteinemia, there is a defect in LDL receptor or Apo B-100. In Type III Hyperlipoproteinemia, there is a defect in Apo E. Hypertriglyceridemia is the most predisposing factor for Acute Pancreatitis.
Tendon Xanthoma means Cholesterol isincreased. Milky plasma means Chylomicrons areincreased. Palmar or Tubero Eruptive Xanthoma means Chylomicron remnant and VLDL
remnant are increased. Deficiency of LCAT leads to increase in nascent discoidal HDL containing Free Cholesterol. Lipoprotein ‘x’ is an abnormal Lipoprotein found in LCAT deficiency and in Cholestatic states. Complete deficiency of LCAT is known as Norum’s Disease, can progress to end stage renal disease. Partial deficiency defi ciency of LCAT LCAT is known k nown as Fish Eye Eye Disease, Dise ase, which is benign condition. condit ion.
LIPID METABOLISM FA synthesis is an Anabolic pathway and occurs occurs in Fed state state and activated activated by Insulin hormone. Major site of Fatty Acid synthesis is Liver. FA synthesis occurs in Cytoplasm. Acetyl CoA (produced in Mitochondria) is the starting material for Fatty Acid Synthesis. RLE of Fatty Acid synthesis is Acetyl CoA Carboxylase (adds CO2 to Acetyl CoAto make Malonyl CoA). Malonyl CoA is the activated form in Fatty Acid Synthesis. Main enzyme of FA synthesis is FattyAcid synthase complex/ Palmitate synthase
complex.
Fatty
Acid synthase is Malonyl-Acetyl a MultienzymeTransacylase, complex consisting of 6 Enoyl enzymatic activities: Ketoacylcomplex synthase, Hydratase, Reductase, Ketoacyl reductase and Thioesterase/Deacylase.
Lipids : 59 59
Malonyl ransacylase
e y ra as a se Enoyl
Releasing enzyme
reductase Ketoacyl reductase
Acetyl ransacylase
ACP Thioesterase
Ketoacyl
synthase
4`-phosphopantetheine
Cys
SH
Subunit division
SH SH
SH Cys
4`-phosphopantetheine
Thioesterase
Ketoacyl synthase
ACP
Acetyl
Ketoacyl reductase
Enoyl
Dehydratase
Malonyl transacylase ransacylase
reductase
Fatty acid synthase multienzyme complex
Fatty Acid Synthase Complex is a dimer consisting of two subunit and each subunit attached to Acyl Carrier protein (has SulThydryl group with Phosphopanetheine). Malonyl-Acetyl Tranacylase enzyme is involved in the loading of Acetyl CoA (2C) and Malonyl CoA (3C). Palmitic Acid / Palmitate is the first Fatty Acid synthesized denovo after 7 cycles of Fatty Acid Synthesis. Synthesis of one Palmitate requires 14 NADPH . Cofactors required for Fatty Acid Synthesis are NADPH, Biotin & ATP. Malate in Cytoplasm gets converted to Pyruvate by Malic enzyme which is a minor source of NADPH.
Citrate Shuttle
60 : Lipids Lipids
Chain Elongation of Fatty Acids occur mainly in ER and also in Mitochondria. Elongation above C10 occurs in ER. Desaturation to produce Unsaturated FA occurs in Smooth ER and mainly in Liver. Enzyme involved in Desaturation is Membrane Bound Enzyme known as Fatty acyl
CoA Desaturase.
Most common Desaturase is Δ9-desaturase. Humans cannot introduce double bonds beyond Δ9 position. Sources ofGlycerol-3-PhosphateareDHAP(byenzymeGlycerol-3-P dehydrogenase) and from Glycerol (enzyme is Glycerol Kinase). Glycerol-3-P Dehydrogenase is present in both Liver & Adipose Tissues. Glycerol Kinase is present only in Liver. Adipose Tissue strictly depends on Glucose Uptake (via GLUT-4) for TG synthesis. RBCs always rely on Glucose because they cannot cannot use Ketone Ketone Bodies Bodies or Fatty Fatty Acids Acids due to Lack of Mitochondria. Ketone Bodies are Acetoacetate, Acetoacetate, β-Hydroxy Butyrate and Acetone. Concentration of Ketone Bodies in Blood (normal person) = 0.2 mmol/L Ratio of β-Hydroxy Butyrate and Acetone in a Normal person is 1:1 wheras in Ketosis it become 6:1. Ketone Bodies can easily cross IMM. Ketone Body Synthesis and Utilization take place in the Mitochondria Ketone Body Synthesis occurs in Liver whereas Utilization occurs in Brain, Heart and Skeletal muscles. Ketone Bodies are synthesized when there is increased Lipolysis and increased Acetyl CoA and decreased Oxaloacetate. Ketone Body Synthesis occur during Starvation and Severe Uncontrolled Diabetes Mellitus. Primary Ketone Body /First Ketone Body synthesized – Acetoacetate. Secondary Ketone Bodies - β-Hydroxy Butyrate and Acetone. Most common Ketone Body found in Blood & Urine is β-OH-butyrate . Acetone is volatilized via Lungs and does not act as a Fuel in the body. It is just released from breath and has diagnosticrole. Rate Limiting Enzyme of Ketone Body Synthesis is HMG CoA Synthase (enzyme common to both Ketone Body Synthesis and Cholesterol Synthesis). Thiolase is a common enzyme for 4 Pathways : KB synthesis, KB Utilization, Cholesterol Synthesis, β-oxidation of FA. Pathways where HMG CoA is Intermediate: KB synthesis, Cholesterol synthesis and Leucine Catabolism. First enzyme of Ketone Bodyutilization is Thiophorase/ Succinyl CoA Transferase – does not use ATP but adds High Energy CoA bond. Energy yield from one Acetoacetate is 19 ATPs. Energy yield from one molecule of β-OH-butyrate is 21·5 ATPs. Rothera’s test is used for detection of KBs and Positive Test indicates Acetoacetate and Acetone.
Positive test indicate Acetoacetate (not Gerhardt’s test – Detection of KBs – Positive Acetone).
No test available for for β -OH-Butyrate.
61 Lipids : 61 Liver cannot use KBs because of the absence of Thiophorase. RBCs cannot use KB because they lack Mitochondria. Neutral Ketone Body – Acetone. Acetone. First enzyme of KB synthesis – Thiolase. Thiolase. Immediate precursor of Acetoacetate – HMG CoA. HMG
Beta Oxidation of FA (odd and even) – in in Mitochondria and occurs in Fasting state or Starvation. ATP produced produced by Oxidation Oxidation of of VLCFA VLCFA and α-Oxidation of FA. No ATP Any defect in β-oxidation leads to Non-Ketotic Hypoglycaemia. Ketotic Hypoglycaemia occurs in Von Gierke’s disease. Carnitine –Acyl Carnitine Translocase enzyme transports Acyl Carnitine into Mitochondrial matrix and it also transports Free Carnitine out from Mitochondrial Matr ix. Muscle does not synthesize FAs. Malonyl CoA (3C) is the Inhibitor of CPT-I (Carnitine Palmitoyl Transferase-I). In Fed state, β-oxidation is inhibited. 106 ATPs are obtained by β -oxidation of 16-C Palmitic acid.
120 ATPs are obtained by β-oxidation of 18-C Stearic acid. 2 ATPs are used in Activation of FA for β -Oxidation. Long Chain Acyl CoA Dehydrogenase :starts breaking a Long Chain FA and can
break upto 12C.
Medium Chain Acyl CoA Dehydrogenase :works after Long Chain Acyl CoA
Dehydrogenase and can do all cleavages below 12C. MCAD deficiency is Autosomal Recessive and is the most common Inborn Error of β-oxidation. During Starvation, patient with MCAD deficiency has low Ketone Bodies, and Hypoglycaemia/Non-Ketotic Hypoglycaemia. Ketotic Hypoglycemia occurs in Von gierke’s Disease and in Alcoholism. JamaicanVomitingSickness occursafteringestionofunripefruitofAkeetreewhich contains a toxin named Hypoglycin. It inhibits Fatty Acyl CoA Dehydrogenase. Generally Fats can never be converted to Carbohydrates but there are two exceptions: Glycerol and Propionic Acid. L-Methyl Malonic Acid and Propionic Acid areexcreted In Vitamin B12 deficiency, L-Methyl in urine. In Vitamin B7 deficiency, Propionic Acid is excreted in urine. Odd Chain Fatty acids can form Glucose (as they form Propionyl CoA) but Even Chain Fatty Acids cannot. β-oxidation of VLCFA occurs in Peroxisomes upto Octanoyl CoA, Rest in Mitochondria. Zelleweger Syndrome/ Cerebro-Hepato-Renal Syndrome occurs because of the accumulation of Polyenoic Acids in Brain (containing >C22).
Zellweger Syndrome is the most severe Peroxisomal Biogenesis Disorder. In Zellweger Syndrome, there is accumulation of Phytanic Acid and VLCFA.
62 : Lipids Lipids
Protein Targeting Disorders are I-Cell Disease, Primary Hyperoxaluria, Familial
Hypercholesterolemia, Zellweger Syndrome & Cystic Fibrosis. Wolman’s Disease is a Lysosomal Storage Disease but not a Sphingolipidoses. Lysosomal Storage Diseases are Mucopolysaccharidosis, Pompe’s Disease, Sphingolipidoses, Sphingolipidose s, Wolman’s Disease, Cystinosis.
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.
X-linked Adreno Leulo Dystrophy/XALD : Defect in the transport of VLCFA
across peroxisomal membrane. α-oxidation of FA occurs in Peroxisomes and ER. In α-oxidation, there is removal of one carbon from Alpha Carbon atom. Refsum’s Disease is defect in Alpha-Oxidation of Fatty Acids and there is accumulation of Phytanic Acid. ω-oxidation of FA occurs in ER. In Omega Oxidation, there occurs occurs oxidation oxidati on of Omega Carbon of Fatty Acid leading leadin g to the formation of Dicarboxylic Acids.
Cholesterol Synthes Synthesis is is Anabolic Pathway and occurs in Fed state and is activated
by Insulin. Insulin. Cholesterol is having four fused Hydrocarbon Rings and it is called Cyclo Pentano
Per Hydro Phenanthrene Ring (Steroid Ring). Cholesterol has 27 Carbons and one –OH group at Position Number 3. All the 27 carbons of Cholesterol are derived deri ved from Acetyl CoA CoA..
Cholesterol is i s required for for Membranes, Steroid Hormones, Lipoproteins, Vi Vitam tamin in D and a nd Bile Acids. Acids.
RLE of Cholesterol Synthesis – HMG CoA Reductase.
HMG CoA Reductase catalyzes the conversion of HMG CoA to Mevalonate. Cholesterol Synthesis occurs in all tissues. Acetyl CoA is starting material and NADPH NAD PH is used for for Reductive Biosynthesis. ATP is also used.
First two steps in Cholesterol Synthesis are same as those in KB synthesis but Cholesterol Synthesis takes place in Cytoplasm. HMG CoA can be converted to Mevalonate, Cholesterol, Ketone Bodies & Acetyl CoA. Compounds derived from Cholesterol are Bile Acids, Vitamin D and Steroid Hormones. Primary Bile Acids are Cholic Acid & Chenodeoxy Cholic Acid. Secondary Bile Acids are Deoxycholic Acid & Litho Cholic Acid. Rate Limiting Enzyme of Bile Acid Synthesis is 7-α Hydroxylase (requires Vitamin C). Ursodeoxy Cholic Acid is a Secondary Bile Acid, used in the treatment of Liver diseases.
CHAPTER MOLECULAR
7
CHEMISTRY & METABOLISM OF NUCLEOTIDES
Nucleic Acid is polymer of Nucleotides. Nucleotide = Nitrogenous Nitrogenous base + Sugar +
Phosphate.
Nucleoside = Nitrogenous base + Sugar.
Purines are Adenine and Guanine. Pyrimidines are Cytosine, Uraciland
Purines Vs Pyrimidines
Thymine.
Thymine presen presentt only in DNA, not in RNA. RNA A, not in DNA. Uracil presen presentt only in RN Thymine is a Pyrimidine Nitrogenous base. Thiamine is a Vitamin (Vitamin B1).
Vitamin B1 has a Pyrimidine like Ring in its structure.
Guanidine is a Protein Denaturating Agent but Guanosine is a Nucleoside of
Guanine (i.e. Guanine + sugar). Most abundant Free Nucleotide in Mammalian cells is ATP. Conjugated Double Bonds in the rings is responsible for Absorption of UV light . 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). AminoAcids absorbs UV-Light at 280 nm. NAD/NADP absorbs UV-Light at 340 nm. Porphyrins absorbs UV-Light at 400nm. Sugar in Nucleic Acids is always a Pentose. This pentose can be Ribose or DeoxyRibose. Ribose exists in Furanose form. At 2’ position, -OH group is present. If oxygen is removed from 2’ position, then it is known as 2’ Deoxyribose. If oxygen is removed from 2’ & 3’ position, then it is known as 2’3’ Di -deoxyribose.
64 : Molecular
NADPH is the ultimate donor of Reducing Equivalents for the conversion of
Ribonucleoside Diphosphate to its Deoxy Ribonucleoside Diphosphate (Enzyme – Ribonucleotide Reductase). Two pathways for synthesis of Purine Nucleotides are De Novo Pathway and Salvage Pathway.
De Novo Pathway is High Energy Consuming Pathway and is most active in Liver.
Phosphoribosyl Pyrophosphate (PRPP) Synthetase catalyze the Synthesis of PRPP from Ribose-5-P. 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. First Purine Nucleotide to be synthesized is IMP (Inosine Mono Phosphate). Parent Purine Nucleotide for AMP and GMP is IMP (Inosine Mono Phosphate). AMP synthesis require Aspartate and GTP. GMP synthesis require Glutamine and ATP. 6-Mercapto Purine is an Inhibitor of Adenylosuccinate Lyase. PRPP is used in Purine Synthesis, Pyrimidine Synthesis, Histidine Synthesis and
Niacin Synthesis. Synthesis.
Salvage Pathway for Purine Synthesis occurs in RBCs,WBCs, Brain and Bone
Marrow.
Inhibitors of Purine Synthesis: PABA(Para-Amino Benzoic Acid) Analogues and
Antifolates.
Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) is enzymeof
Salvage Pathway which converts Hypoxanthine to IMP and Guanine to GMP. Complete deficiency of HGPRT leads to Lesch Nyhan Syndrome (Gout & Self
Mutilation). Partial deficiency of HGPRT leads to Kelley Seegmiller Syndrome (only Gout). Adenine Phospho Ribosyl Transferase (APRTase) converts Adenine to AMP. Adenosine is the only Purine Nucleoside which can be salvaged i.e. Adenosine gets
converted to AMP by Adenosine kinase. 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. Increased ADA levels is highly suggestive of Tuberculosis . Deficiency of ADA leads to SCID (Severe Combined Immuno Deficiency) Adenosine (substrate) is increased which gets converted to Deoxy ATP, which is inhibitor of Ribonucle Ribonucleotide otide Reductase. So, synthesis of Deoxyribonucleotidesstops & Cell cannot divide di vide due to lack of DNA Synthesis. Synthesis. SCID was the first disorder to be treated by Gene Therapy. Xanthine Oxidase: Rate Limiting Enzyme of Purine Catabolism. Xanthine Oxidase (RLE) catalyzes the conversion of Xanthine to Uric Acid and requires Molybdenum. Allopurinol is the Suicide Inhibitor of Xanthine Oxidase.
Molecular : 65 65 Catabolic End Product of Purines in Primates, Amphibians, Reptiles and Birds is Uric Acid. In Non-Primate Mammals, Uricase /Urate Oxidase forms Allantoin as the End Product of Purines. Combined Mechanism of Hyperuricemia (increased synthesis of Uric Acid and
decreased excretion): Von Gierke’s Disease, Aldolase B Defic Deficiency, iency, Shock and Alcohol. HGPRTDeficiency Increased Uric Acid. Acid. Xanthine Oxidase Deficiency Decreased Uric Acid. PRPPSynthetasee Deficiency Decre PRPPSynthetas Decreased ased Uric Acid. Acid.
Low Purine Diet is Milk (specially cow’s milk), Yoghurt, Cheese and Vit C - help in
excretion of Uric Acid from Kidneys. High Purine Diet is Spinach, Mushrooms, Peas, Cauliflower, Meat, Fish (tuna), Liver. Gout Patients should avoid Heavy exercise & Alcohol as it leads to Lactic Acidosis & Hyperuricemia. First Pyrimidine Nucleotide tobesynthesizedis OMP (Orotidine Mono Phosphate).
Rate LimitingSynthetase-II). Step of Pyrimidine Synthesis in Eukaryotes is CPS-II (Carbomoyl Phosphate Rate Limiting Step of Pyrimidine Synthesis in Prokaryotes is Aspartate Trans Carbamoylase. OPRT and Decarboxylase are Bifunctional Enzymes (two enzyme activities on a single protein). This enzyme/protein is known as UMP Synthase. UTP gets converted to CTP by enzyme CTP Synthetase (requires energy from ATP) and requires Amino group from Glutamine. Orotic Aciduria is a rare Autosomal Recessive Disorder, which is defect in UMP Synthase. Orotic Aciduria is the most common Metabolic Error in Pyridimine Synthesis. In Type I Orotic Aciduria, both OPRT and Decarboxylase enzymes are deficient. In Type II Orotic Aciduria , only Decarboxylase is deficient.
Megaloblastic Anaemia occurs in Orotic Aciduria due to deficiency of Pyrimidines. 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. UDP-Glucuronic Acid is used for the Conjugation Reactions (like Bilirubin), Synthesis of Proteoglycans. PAPS (Phospho Adenosyl Phospho Sulphate) act as Sulphate Donor. Cyclic AMP and Cyclic GMP act as Second Messengers. NAD, NADP, FAD, NADP, FAD, FMN act as Coenzymes. DNA and RNA are Nucleic Acids (Polymer of Nucleotides). Hershey & Chase Experiment was done on Bacteriophages and it proved that DNA is the Genetic Material in organisms. DNA is Double Stranded in both Eukaryotes and Prokaryotes.
In DNA, Purines always equals Pyrimidines. RNA is Single Stranded, so Purines are not necessarily equal to Pyrimidines.
66 : Molecular DNA is Helical (right handed) and the two strands are Antiparallel. DNA is made up of four Nitrogenous bases : Adenine (A), Thymine (T), Guanine (G) and Cytosine (C). B-DNA and A-DNA are Right Handed whereas Z-DNA is Left Handed. B-DNA is the major DNA in cells.
Z-DNA has a function in regulation of Gene Expression, particularly in GC sequence
(alternating Purine-Pyrimidine). Gene Expresssion = Transcription +Translation. Thymine (T) is present instead of Uracil (U) in DNA to prevent Mutations. Cytosine gets converted to Uracil (by removal of amino group) very spontaneously Deaminase se. in DNA, by enzyme Cytosine Deamina Main difference between DNA and RNA is Sugar i.e always Deoxyribose in DNA and its always Ribose in case of RNA.
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 Pyrimidin Pyrimi dines es equal (C+T)tobut the it number of (A+T) is not (G+C), can be variable. Nucleosomes = DNA +Proteins. DNA is negatively charged due to Phosphates. Prokaryotes have circular double stranded DNA whereas Eukaryotes have linear double stranded DNA. Histone Octamer contains H2A, H2B, H3 and H4 Histone proteins. Linker / Spacer DNA is present in between two Histone Octamers. It is associated with Linker Histones i.e H1 and H5.
Histone Proteins have positive charges because they are rich in Basic Basic Amino Acids Acids (Lysine, Arginine). Due to opposite opposite charge on DNA and Histone Proteins, they get tightly bound forming Nucleosomes. transcriptionally nally active. If DNA is separated from Histone, then it is free or is transcriptio 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 GeneExpression. Various ways of Histone Modifi Modifications cations are: Acetylation, Phosphorylation, Methylation, ADP Ribosylation, Mono-Ubiquitylation and Sumoylation.
Molecular : 67
Acetylation and Phosphorylation of Histones lead to increased Euchromatin Formation Gene Activation. Deacetylation and Dephosphorylation of Histones leads to Gene Inactivation. Histone Methylation leads to GeneInactivation. DNA present in compact form along with Histones is known as Chromatin and
exists only in Eukaryotes. During Mitosis, Chromatin is heavily condensed but during Interphase, it is less condensed. Euchromatin is loose DNA and genes are active. Heterochromatin is tightly bound DNA and genes are inactive. The interconversion of Euchromatin and Heterochromatin is known as Chromatin Remodelling. Proteins and DNA are bound by Weak bonds such as Hydrogen, Ionic, Vanderwaals and never by Covalent bonds. The bonds present in DNA are Hydrogen bonds, 3’ 5’ Phosphodiester bonds and β-N-Glycosidic bonds. 3’ 5’ Phosphodiester bonds are formed between 3’OH of previous Nucleotide with 5’ Phosphate of incoming Nucleotide. At 5’end of a Nucleic Acid, there is always a Free Phosphate present present.. At 3’end of a Nucleic Acid, a Free –OH is always present. New Nucleotide is alway Nucleotide alwayss added at the 3’end of a Nucleic Acid. Synthesis always occurs in 5’ to 3’ direction. Reading & Writing of Base Sequence is always done in 5’ to 3’ direction. Nucleases are enzymes which break the Phosphodiester bond present between the Sugar and Phosphate in a Nucleic Acid. Nucleases are of two are two types: types: Exonucleases and Endonucleases (or Exci-Nuclease). Exonuclease cut the Nucleic Acid from the sides either from 5’ end or from 3’ end. 5’ 3’ Exonuclease starts cutting from 5’end and moves from 5’ to 3’ direction. 5’ 3’ Exonuclease starts cutting from 3’end and moves from 3’ to 5’ direction. Restriction Endonu Endonucleases cleases (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 . Pallindromes are same sequences present on opposite strands of DNA, read in 5’ 3’ direction. DNA Methylation at CG sites is the most common method of Gene Inhibition in Genomic Imprinting. In mammals, 70-80% of CpG Cytosines are Methylated. CpG Islands usually has G+C content greater than 50%. Majority of Gene Promoters have high CG content. In Non-Hematopoetic cells, DNA Methylation is done to inhibit Transcription of Globin chain genes.
In
, genes areHypomethylated. Hematopoetic cellsGenome Diploid Human The consists of 23 pairs of Chromosomes (composed of 20-25 thousand genes).
68 : Molecular Haploid set of Chromosomes is estimated to be 3·2 × 109 Base Pairs. Genes consisting of DNA base pairs are located on Chromosomes. A Gene is a sequence of Base Pairs that produces a functional product including RNA molecule and subsequently a Peptide.
The Diploid Genome contains two alleles of each gene. An Allele is positioned on a locus (specific location locati on of a gene or DNA DNA sequence on a chromosome).
Chromosomes 1 to 22 are called Autosomes and the 23rd pair is Sex Chromosomes i.e. X and Y.
Homologous Chromosomes
are Chromosome Pairs (one from each parent) that are similar in Length, Gene Position, and Centromere Centrome re location. location. The position of genes on each
Homologous Chromosome is exactlysame, however, the genes genes may contain differentalleles.
Sister Chromatids are exact copy of each other.
Centromere is aconstricted point of of the chromo chromosom some. e.
Telomeres aree areends nds of chromosomes.
Exons are the genes ge nes which give rise to proteins.
Introns are Intervening Sequences which are present in between Exons. Exons.
Single Nucleotide Polymorphisms (SNPs) is the most common type of
Polymorphism in which Single Nucleotide is variable in different population.
Repeat Length Polymorphism: Tandem repeats of a particular length DNA occurs
and the number of these repeats are variable in different population. Repetitive Sequence in DNA (Satellite DNA) are often clustered in Centromeres & Telomeres.
Short Tendem Repeats/STRs are known as Microsatellites and the repeat size is
2-6 base pairs.
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
69 Molecular : 69 high frequency frequen cy during each round of Mitosis. So Tumor Cell gives a different Genetic Fingerprint than th an the host cell. Variable number of Tandem repeats/VNTRs are also known as Mini-Satellites and the repeat size is 15-70 base pairs.
Mitochondrial DNA: same like Prokaryotic
DNA i.e. Circular double stranded and no Introns present. Mitochondrial (DNA/mtDNA) is
-Polymerase (gamma). synthesized by γ -Polymerase mtDNA has high rate of Mutation as compared to Nuclear DNA.
mtDNA contains 16500 base pairs and 67 genes, all of which are essential for normal Mitochondrial function.
Thirteen of these 67 genes prov provid idee
instructions for making enzymes involved in oxidative phosphorylation. AUA Codon codes for Isoleucine but in mitochondrial DNA , it codes for Methionine.
UGA Codon is astopcodon butinmitochondrial DNA, it codesfor Tryptophan. AGA& AGG codes for Arginine butinmitochondrial DNA, these are Stop Codons. Mitochondria Mitochondriall inheritanc inheritancee is Non-Mendelian & it is only transmitted through
Mother. Mutations in mt DNA mostly affect Oxidative Phosphorylation and there is decreased ATP in cells.
Diseases related to Mutation Mutationss in mtDN mtDNA: A: MELAS, Kearns syndrome, Leber
Hereditary Optic Neuropathy, Leigh syndrome & NARP syndrome.
MELAS : Mitochondrial Encephalopathy, Lactic Acidosis and Stroke like Episodes. Kearns/Sayre Syndrome : Large deletion of mt DNA. Patient has Ophthalmoplegia and Retinopathy.
Leber Hereditary Optic Neuropathy : Optic Nerve Nerve affected leading to Vision Loss. Loss. Leigh Syndrome : Brain affected leading to Developmental Delay, Delay, Muscle Weakness. Neuropathy pathy,, Ataxia & Retinitis Retinitis Pigmentosa. Pigmentosa. NARP Syndrome : Neuro
DNA Recombination Molecular Cloning is
or
the insertion of DNA fragments to produce new Nucleotide sequence arrangements.
Recombination DNA
+
70 : Molecular
Types of DNARecombination:
Homologous Recombination (HR) Site Specific Recombination (SSR) or Non-homologousrecombination Transposition 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. Examples of Site Specific Recombinase are CRE Recombinase, λ Phage encoded INT protein , Yeast Yeast FLP FLP Recombinase Recombinase CRE/Lox Recombinase associates specifically with the Lox P locus. 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. Transposable Elements are known as Jumping Genes as these are DNA sequences that can move from one chromosome locus toanother.
There are two types of TransposableElements: Transposons Retrotransposons Transposons move by means of DNA Intermediate. Retro Transposons move by means of an RNA Intermediate. Small Scale Mutations (Micro Alteration) or Gene Mutation are Point Mutations, Insertions and Deletions.
Point Mutations involve Transition and Transversion. Transitions - Pyrimidine to Pyrimidine or Purine to Purine Transversions - Pyrimidine to Purine or Purine to Pyrimidine Each Aminoacid has morethan one codons, isknown as Degeneracy or Redundancy.
Point mutations are of three types namely Silent, Missense and Non Sense Mutations. In Silent Mutation, Primary Structure of Protein always remain the same. Codon is replaced by another codon which codes for same Amino Acid. Silent Mutations occur due to Degeneracy of Codons (codon is changed but Amino Acid is not changed). So Degeneracy prevents Mutations. In Non Sense Mutation, Primary Structure of Protein will be changed, as there will be premature premature Termination Termination of of Protein Synthesis Synthesis due due to introductio introduction n of Stop Codon. Codon. In Missense Mutation, a Codon is replaced by another Codon coding for a different Amino Acid. 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.
Diseases due to Trinucleotide Repeat Expansion:
Hungington Disease Fragile XSyndrome
Frame Shift Mutation Splice Site Mutation
Molecular : 71 71
Huntington Disease: Neuro Neurogenerat generative ive - Trinucleotide Trinucleotide Repeat Expansion Expansion of Codon
for Glutamine, leads to formation of an abnormal Protein which is degraded easily producing Toxic Fragments Fragments that aggregate aggregate in Neurons. Neurons. Fragile X Syndrome: Trinucleotide Repeat Expansion leading to Hypermethylation which cause Gene Silencing.
Frame Shift Mutation: Addition or Deletion of Nucleotides (not divisible by 3) alter the reading readin g frame. frame.
Splice Site Mutation: Can interfere in the removal of Introns from the Primary
Transcript.
Large Scale Mutations (Macro alteration) or Chromosomal Mutations include
Amplification/Duplication, Deletions, Inversions, Translocations and Aneuploidy. An Inversion that involves the Chromosome’s c onstriction point (Centromere) is called a Pericentric Inversion. 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. Translocation occurs when a piece of one chromosome breaks off and attaches to another chromosome. It can be Balanced or Unbalanced. If no genetic material is lost or gained from the Chromosome, then called Balanced Translocation. This occurs between the Homologous Chromosomes. If there is a gain or loss of genetic material from the chromosome. Then called
Unbalanced Translocation. In Robertsonian Translocation, there is joining of long arms of two chromosomes. The tiny short arms are usually lost. Aneuploidy is abnormal Chromosome Number. It can be Trisomy (three chromosomes instead of two) or Monosomy (one chromosome).
DNA REPLICATION
Central Dogma of Molecular Biology: Theflow of information from DNAto RNA
and then to Proteins.
Reverse Transcription is the synthesis of DNA from RNA by enzyme RNA Dependent DNA Polymerase. Reverse Transcription occurs in Retroviruses such as HIV virus, Transposons and
Telomerase.
DNA Replication and Synthesis of Histones occurs in S-phase of Cell Cycle. DNA Replication is Semi-Conservative in nature (one parental strand is conserved
each time). Prokaryotes have one Origin of Replication (ori) while Eukaryotes have multiple ori sites. Ori is AT rich sequence and can be separated easily. DNA Replication is bidirectional (Replication moves in both directions from the ori). The Temperature at which half of the Helical Structure is lost is known as Melting Point (Tm).
72 : Molecular
High GC content of DNA raises Tm. Enzymes of DNA Replication: Helicases, Topo-isomerases, SSBs (single strand
DNA Binding Proteins), Primase, DNA Polymerase III, DNA Polymerase I, and DNA Ligase.
Helicases cause strand separation using ATP and creates Positive Supercoils. Overwinding is known as PositiveSupercoiling . Underwinding is known as Negative Supercoiling. 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 onestrand. Type II Topo-Isomerases cut two strands. DNA Gyrase is a counterpart of Topo-Isomerase-II in Prokaryotes. This enzyme enzyme
uses ATP and introduces Negative Supercoils.
Helicases and Topo-Isomerases work in tandem to do strand separation. 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 Nucleas es attack. These These are not Enzymes.
RPA (Replication Protein A) In Eukaryotes, for prevention of Reannealing. Helicase, Topo-Isomerases and SSBs Together known as Unwinding Proteins. Primase synthesize Primers (RNA) from Template DNA. DNA Dependent RNA Polymerase in Replication is Primase. In Eukaryotes, α-Polymerase synthesize primers. In Prokaryotes, DNA-G protein synthesizepr synthesizeprimers imers.. The Template or the Parent strand is 3' read from 3’ to 5’ direction. 5'
The strand synthesized in one stretch is known as Leading strand. The strand synthesized in short
Leading strand
fragments (Okazaki fragments) is Lagging known as Laggingstrand. strand Only one Primer is needed need ed for 3' Leadingstrandbut multiple Primers 5' are needed for Lagging Strand.
3'
5' 3
5' Okazaki fragments fragments
DNA Polymerase III synthesize both Leading and Lagging strands. Substrate for this enzyme is Deoxyribo Nucleotide Triphosphates (dNTPs). DNA Polymerase III has Polymerase (5’ 3’ Polymerase) Activity & 3’ 5’ Exonuclease (Proofreading Activity). DNA Polymerase III synthesizes OkazakiFragments. DNA Polymerase II has 3’5’ Exonuclease (Proofreading Activity) & Repair .
DNA Polymerase I fills the gap between Okazaki Fragments.
If 2’3’ Dideoxy Ribose is used, DNA synthesis stops.
Molecular : 73 73
If any of the four substrates i.e dATP, dGTP, dCTP, dTTP is not available, DNA synthesis stops.
DNA Polymerase I remove RNA Primers from both Leading and Lagging strand. DNA Polymerase I has Polymerase (5’3’ Polymerase) activity and 3’ 5’
Exonuclease (Proofreading Activity) & Exonuclease Activity. 5’ 3’ Klenow Fragment is a fragment of DNA Polymerase Iwhich has 5 3’ Polymerase Activity and 3’ 5’ Exonuclease Activity.
DNA Ligase acts only on the Lagging strand and creates 3’5’ Phosphodiester bond
using ATP.
Telomeres have tandem repeats of a transcriptionally inactive Hexameric sequence (TTAGGG)n.
Telomeres provide structural support to the Chromosome, prevent attack by Nucleases Nucleas es and distinguis distinguish h a true end of Chromosom Chromosomee from break in dsDNA. dsDNA.
Telomeres shorten as cell divides. Telomerase enzyme provides longevity tocells.
Telomerase is Ribonucleoprotein i.e. it has RNA + Protein.
Telomerase is not a Ribozyme because becau se this RNA is not used use d as enzyme. enzyme. It is used use d as a template for for DNA synthesis.
Telomerase is RNA Dependent DNA Polymerase or Reverse Transcriptase. Telomerase activity increases in Cancer and decreases in Aging.
Germ cells have more Telomerase Activity than Stem Cells. Proofreading is correction during Synthesis, occurs in S-phase. DNA Repair is correction after Synthesis. Proofreading has 3’5’ Exonuclease Activity. Proofreading and DNA repair both are done by DNA Polymerase II in Prokaryotes. In Eukaryotes, all Enzymes can do Proofreading except α and β-Polymerase. Most Repairs occur in G1 phase of Cell Cycle. But Mismatch Repair occurs in G2 phase. DNA Repair is done mainly by β-polymerase in Eukaryotes. ε-polymerase has a minor role in DNA repair. Also has role in Leading Strand
synthesis in case of Eukaryotes.
δ-Polymerase in Eukaryotes – Lagging Lagging Strand Synthesis.
74 : Molecular Compa Comparisonof risonof prokaryo prokaryoticandeukaryotic ticandeukaryotic DNA polymerase polymerase E. coli
Eukaryotic
Function
I
II
β γ
III
ε δ
DNA-G
α
Remove primer and fill the gap
Remove primer and fill the gap DNA repair Microchondrial DNA synthasis Leading strand synthesis Lagging strand synthesis
Primase
Primers are removed by RNase, FEN-1 (Flap Endonuclease) and δ-Polymerase (minor role).
Norfloxacin, Ciprofloxacin inhibit DNAGyrase. Arylhydrazinopyrimidines inhibit DNA Polymerase III.
inhibit Enzymes of DNA
Ethylmaleimide, Aphidicolin, Butyl-Phenyl-dGTP Replication.
Didanosine (2’3’dideoxyinosine), Cytarabine, Vidarabine, Zidovudine, Acridine, Ethidium, Actinomycin, Mitomycin are the inhibitors that directly interact with DNA.
TRANSCRIPTION & TYPES OF RNAs
There are three types of RNAs :
mRNA rRNA tRNA
mRNA is the most Heterogenous RNA, synthesized in Nucleolus.
rRNA (ribosomal) is the most abundant and is synthesized in Nucleolus. tRNA (transfer) is the smallest and has maximum Modified Bases e.g. Pseudouracil. Both mRNA and tRNA are synthesized in the Nucleus.
Molecular : 75 75 tRNA and rRNA have uncapped5’ ends and no Poly A tail at 3’ends. tRNA and rRNA are not translated. tRNA is the Adaptor to carry specific Amino Acids. tRNA has T-arm/TΨC arm/ Pseudouridine arm which helps in non covalent binding of A-site of ribosome during initiation and elongation. DHU arm/D arm/Dihydrouridine arm selects the correct amino acid byrecognizingspec byrecog nizingspecific ificAminoAcyl tRNA Synthetase enzyme. Codon is present on mRNA (messenger) and Anticodon is present on tRNA. tRNA.
Amino acid is covalently attached at the 3’end of tRNA. mRNA which is formed from more than one gene is known as Polycistronic andis a characteristic of Prokaryotes. In Eukaryotes, each mRNA comes from a single gene knwn as Monocistronic
mRNA. Bacterial/ Prokaryotic Ribosome: 70S comprising larger subunit 50S (5s, 23s rRNA) and smaller subunit 30S (16srRNA). Eukaryotic Ribosome: 80S comprising larger subunit 60S (5s ,5.8s, 28s rRNA) and smaller subunit 40S (18srRNA). Exons or Coding DNA sequence are only 1-2% of all Genome. Rest is Non-Coding. Presence of Introns prevent Mutations.
Introns are transcribed and not translated.
Only eukaryotic DNA have introns. Coding RNA is only mRNA . Rest are Non Coding RNAs. The final product of Gene Expression can be a Protein or RNA, depending on the Gene.
Ribozyme mean meanss RNAactas RNAac tas Enzyme. Enzyme. Substrate of Ribozyme is mostly RNA.
Ribozyme catalyzes the breakage and synthesis of Phosphodiester Ribozyme Phosphodies ter bond and noATP is used. Telomerase and RNase H are not Ribozymes.
Ribozymes : rRNA, Splicing Ribozyme (snRNA), Ribonuclease P. Ribonuclease P cleaves tRNA. Transcription is the synthesis of RNA from DNA by enzyme RNA Polymerase
(DNA Dependent RNA Polymerase).
76 : Molecular Transcription has higher error rate thanReplication.
RNA Polymerase does not require
Primer and cannot do Proofreading.
RNA polymerase polymerase
RNA Polymerase activity in 5’3’
direction.
Holoenzyme of
RNA
Polymerase
contains 5 subunits- σ ααββ’ω. α, ω subunit is required for Enzyme Assembly.
β subunit has Catalytic Activity.
β’ subunit is required for Template binding and σ for Initiation/Recognizing
Promoter.
ααββ’ω make the Core Enzyme represented as ‘E’. E + σ subunit makes the Holoenzyme. Prokaryotes have single RNAPolymerase.
TypesofRNAPolymeraseinEukaryotes: TypesofRNAPolymeraseinEukaryotes: 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
Template Strand Strand where gene is present, also known as Anti-Sense/ Non-
Coding/ Minus strand.
Non Template Strand also known as Sense/ Coding/ Plus strand. RNA Polymerase first binds with the Promoter Region (helps in initiation but
promoter promo ter region is not transcrib transcribed). ed).
TATA box in Prokaryotes known as Pribnow Box presen presentt at -10 position on DNA
(10 base pairs upstream to the transcription startsite).
Molecular : 77 TATA box in Eukaryotes known as Hogness box presen presentt at -25 position on DNA (25 base pairs upstream to the Transcription Start Site).
-35 sequence present only in Prokaryotes, 35 base pairs Upstream to the
Transcription Start Site.
CAAT box /-75 sequence in Eukaryotes, present 75 base pairs Upstream to
Transcription Start Site.
Regulatory Sequences are present on coding strand in 5’ →3’ sequence. The first base at Transcription Start Site is given +1 number. There is no base
designated as ‘0’. The bases to the left or towards 5’end are assigned negative number or called Upstream. Substrate for Elongation step in Transcription is Ribonucleotide Triphosphates. Two high energy Phosphates used to add one Ribonucleotide . Termination of transcription may be Rho- dependent (ρ) or Rho -independent . Termination n requires ρ-protein and uses ATP and Helicase activity. Rho-dependent Terminatio It releases the RNA at Termination Site.
Inhibitors of Transcription are Actinomycin D, Rifamycin and α -Amanitin. Enhancers of Transcription increase the rate of initiation and can be located
upstream or downstream of promoter. Three Post Translational Modifications occur in all eukaryotic mRNA. These are also known as RNA processing. These are a re Cap at 5’end, removal of Introns/ Splicing and Tail at 3’end. 7-Methyl Guanosine Cap is added at 5’end of mRNA by Guanylyl transferase by unusual 5’5’ Triphosphate Linkage. Methyl group isdonatedbySAM byenzy byenzyme me Guanine 7 methyltransferase (occurs in cytoplasm). Alternate Splicing and RNA Editing are exceptions to One Gene One Protein theory. Cap facilitates the initiation of Translation and stabilizes mRNA by preventing it from attack by 5’ Exonucleases.
78 : Molecular
At 3’end, a Poly A Tail is added by enzyme enzyme Polyadenylate Polymerase. Polymera se. SplicingRibozyme Self SplicingIntronsandSpliceosomal SplicingIntronsandSpli ceosomal Introns. ntrons.
Immature mRNA has both Intron & Exon.
only Exons Mature mRNA contains Lariat Formation occurs in Group II
Self Splicing Introns and Spliceosomal Introns.
snRNA (small nuclear) which takes part in Splicing U1,U2,U4,U5,U6. snRNA have unique 5’ caps. Sm-class snRNA have 5’ Trimethyl Guanosine Caps, & Lsm-class snRNA have 5’ Monomethyl Phosphate caps. Differential RNAProcessing/ RNAEditing is a mechanismtoproduce diverseset
of Proteins from a limited set s et ofGenes. Transcription and Post Transcriptional Modifications occur in Nucleus but in case of rRNA, they occur in the Nucleolus. 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. RS are cis-acting if they control Gene Expression of a Gene that lies on the same chromosome as the RS. RS are trans-acting if they are produced from a particular Gene from a Chromosome but they affect affect the Regulation Regulation of a Gene Gene which which is lying lying on the other other Chromoso Chromosome. me. Protein Motif is that modified portion of a Regulatory Protein which binds to Response Element on DNA. Response Element is a DNA sequence which gives a site of Protein Binding. At this site of DNA, Protein Motifbinds. Helix Turn Helix Protein Motif: Regulate genes of development. Zinc Finger Protein Motif : e.g. Steroid Hormone Receptors. Helix Loop Helix Protein Motif: Regulate Immune System Genes.
Leucine Zipper: Regulate Cell Division. OPERON MODEL Operator is a site on DNA that regulate the activity of the Gene by binding a Protein
known as Repressor. Inducer can bind to Repressor and can remove remove Repressor from Operator site leading to Gene Activation. Inducer is Lactose Lactose in Lac Operon. Operon means a complete unit operating on itself. It has the capability to activate or inhibit the Genes. Operons are found in Prokaryotes . O-site : Operator lies downstream to Promoter. CAP site : lies Upstream of Promoter. CAP-cAMP Complex (trans acting protein) binds here.
Lac Operon Genes are activated if glucose decreases in the environment of E.Coli and if Lactose is present.
Molecular : 79 79
Lac I (Inhibitory) is a House Keeping Gene i.e. always active and has its own
Promoter. Lac I produces Repressor Tetramer (a trans acting factor). Lac Z codes for β-Galactosidase – breaks Lactose to Galactose and Glucose. breaks Lac Y codes for Permease – Facilitate permeation of Lactose into the cell. Facilitate
Lac X codes for Transacetylase - Acetylates Lactose.
TRANSLATION Codons are Nucleotide Triplets (3 nucleotides make 1 codon). 4 Nucleotide Bases used to produce three base Codons i.e. A, U, C, G. Total number of Codons possible =64. STOP Codons : UAA, UAG, UGA – do not code for any Amino Acid. do Methionine and Tryptophan have only 1 Codon i.e. do not show degeneracy. Genetic Code is Unambiguous : each Codon is specific for its Amino Acid e.g. AUG will always Code for Methionine. Genetic Code is Universal i.e well conserved during evolution with only slight differences. Genetic Code is Non-Overlapping and Commaless.
There is Antiparallel binding between Codon Codon and Anti-Codo Anti-Codon n in tRNA. tRNA. Wobble Hypothesis: Movement of first base of Anti-Codon can occur to allow nontraditional base pairing with the last base of Codon. This movement is called Wobble, which allows single tRNA to recognize more than one Codon. Ribosomes are spherical bodies made up of RNA and Proteins. They are not considered Organelles as they are Non-Membranous. Mitochondria contain their own Ribosome (55s) and their own unique circular dsDNA. mRNA is translated in 5’3’ direction and Proteins are synthesized from Amino terminal to Carboxy terminal. Activation of Amino Acids or Charging of tRNA to make Aminoacyl tRNA is done by enzyme enzyme Amino Acyl tRNA Synthetase. The enzyme Amino Acyl tRNA Synthetase is the only point of Proofreading in Translation. It is responsible for Fidelity/Accuracy of Protein Synthesis. Amino Acyl tRNA Synthetase has 20 Isoenzymes, one for each Amino Acid.
80 : Molecular
Hydroxy-Proline a derived Amino Acid does not require Amino Acyl tRNA
Synthetase. Initiation Codon is AUG which Codes for Methionine in Eukaryotes and N-Formyl Methionine in Prokaryotes.
eukaryotes 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 fac factor tor EF - Elongation factor eEF - eukaryotic Elongation factor RF - Releasing factor eRF - eukaryotic Releasing factor
For Initiation in Eukaryotes, ATP and GTP are required. For initiation in Prokaryotes, no ATP and GTP are required and Initiation Factors IF-1, IF-2 and IF-3 are required. ShineDalgarno Sequence (SDsequence) ispresentin Prokaryotes on mRNAwhich is Purine rich and has complementary bases with 16s rRNA, present 10 base pairs Upstream to the Initiation Codon. P- site/Polypeptide Site : always lies on left side and Polypeptide is released from this site. A- site/Acceptor Site : All Amino Acids are accepted here EXCEPT first Methionine. In both Prokaryotes and Eukaryotes, N- terminal Methionine is removed before Translation is completed. Peptidyl Trans Transferase ferase releases Polypeptide from P-site. It has Role in Elongation and Termination. Addition of Amino Acid tothe Carboxyend of growingchain isknownas Elongation. Small ribosomal unit binds mRNA and determines the accuracy of Translation by correct Base Pairing. Large Ribosomal Unit catalyze formation of Peptide Bond. Releasing factors (RF1, RF2, RF3) recognizes Stop Codons in Prokaryotes. GTP is required for Termination in both Eukaryotes and Prokaryotes. 2 ATP (activation (activati on 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 pol ypeptide chain. Both Transcription and Translation occur in Cytoplasm in Prokaryotes. In Eukaryotes, Transcription occurs in Nucleus and Translation in Cytoplasm and hence a discontinuous process.
occurs in inhibit casebit ofInitiation Selenocysteine and Pyrrolysine. Co-Translational Modification Streptomycin binds 30S-50S interfac interface e and inhi In itiation.. Chloramphenicol inhibits Peptidyl Transferase.
Molecular : 81 81
Tetracyclin binds to 30S subunit subunit of Ribosome. Ribosome. Erythromycin inhibits Translocation. Dipthera toxin inhibits EF-G andEF-2. Post Translational Modifications e.g. Covalent Modification phosphorylation,
Glycosylation, Hydroxylation, Acetylation, Vit K Dependent Carboxylation of Glutamate, Biotin covalently bound to Carboxylases.
Glycosylation is the most frequent Post Translational Modification of Protein. Non-Enzy Non-Enzymatic matic attachment attachment of of Sugar to Protein Protein is known known as Glycation .
TECHNIQUES IN MOLECULAR BIOLOGY
Samples taken for PrenatalDiagnosis: 1) Amniotic fluid : know known n as Amniocentesis Amniocentesis – – done done after 16 weeks of Gestation. 2) CVS (Chorionic Villus Sampling) – done done after 11 weeks of Gestat Gestation. ion. 3) Fetal Blood Sampling : done after 18 weeks of Gestati Gestation. on. Blood (WBCs), Cheek Swab, Adult samples which can be taken for DNA testing – Hair.
Epigenetics is information in the Genome which alter thepattern of Gene Expression.
This is Chemical Modification of DNA or Proteins but is not change in DNA Code. Epigenetic Changes are transferred to next generation and are reversible . Epigenetic Changes are : DNA Methylation, PTMs of Histones (occurs mostly on lysine residue on proteins) and changes in high order of chromosome structure.
DNA Methylation of Cytosine of CG sites by Enzyme – DNA Methyl transferases (DNMTs). Acetylation of Histone by enzyme HAT (Histone Acetyl Transferase). Deacetylation of Histone by enzyme HDAC (Histone Deacyetylases). Histone Methylation can be done by Histone Methyl Transferases. Epigenetic changes causes diseases such as Cancer, Aging and Fragile X-syndrome. 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. 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. Genetic Anticipation : The number of repeats increases from generation to generation until affected individual is produced.
82 : Molecular Epigenetic Changes regulate Gene Expression, inactivate X-chromosome and has a role in Genetic Imprinting and Defence Mechanism. Euchromatin is transcriptionally active. Heterochromatin is transcriptionally inactive. Facultative Heterochromatin means the Chromatin has ability to become transcriptionally active again. E.g. X-chromosome Inactivation in Female Mammals. Constitutive Heterochromatin is always inactive andrepetitiveand usuallypresent in Centromere & Telomere and provides structural role. Genomic Imprinting is an Epigenetic Phenomenon which results in Mono-Allelic Expression i.e. allele from one parent is expressed (either Maternal or Paternal). Paternal Imprinting : means Paternal Allele is inhibited and only Maternal Allele is functioning. Maternal Imprinting : means Maternal Allele is inhibited i nhibited and only Paternal Pat ernal Allele Allele is functioning. Most DNA Methylation is removed at Fertilization and re-established during Embryogenesis. Mechanisms of Genomic Imprinting: DNA Methylation at CG sites, Histone
Acetylation and Histone Deacetylation.
Detection of GI: Na Bisulphi Bisulphite te Method, Method, ChIP, ChIP-S ChIP-Sequencing equencing,, Flourescent Flourescent In
Situ Hybridization and Methylation-Sensitive Restriction Enzymes. Na Bisulphite Method detects DNA methylation. X-Chromosome Inactivation in females is correlated with extensive CG Island Methylation. Alterations in Gene and CG Island Methylation Patterns are seen in Aging and in Cancer. Chromatin Immunoprecipitation (ChIP) detects Protein-DNA Interactions and Post-Translational Modifications of Histones. Microarray,, but ChIP is Chromatin Immunoprecipitation. chip is Microarray ChIP-on-chip/ChIP-chip Technique combines Chromatin Immunoprecipitation with DNA Microarray.
ChIP-chip can detect Protein-DNA interactions invivo. RNA Immunoprecipitation (RIP) and Cross-Linking Immunoprecipitation (CLIP)
detects binding of Protein to RNA. RIP uses Formaldehyde-Cross Linking to induce covalent attachment of Proteins to RNA. CLIP uses UV-Cross Linking to induce covalent attachment of Proteins to RNA. UV-Cross Linking is irreversible and more specific i.e bind RNA with Proteins that are in close proximity. Components of PCR (Polymerase Chain Reaction): ds-DNA, two Primers, Thermostable Polymerase - e.g. Taq Polymerase, Deoxy-Ribonucleotides and Mg ++. PCR involves Denaturation (Strand Separation), Annealing (Primer Addition) and (Synthesis). These cycles cycles of Denaturation, Annealing Anne aling and Elongation are Elongation repeated again and again to synthesize large la rge amount amount of DNA. DNA.
Molecular : 83 83
Thermostable DNAPolymerase enzymes with Proofreadingfunction in PCR: Pfu (Pyrococcus furious), Pwo(Pyrococcus woesei) Polymerase and Tli (Thermococcus litoralis) /Ven /Ventt Polyme Polymerase. rase. Best thermostable DNA Polymerase enzyme with Proofreading function is Pfu Polymerase.
Pfu is used in conjunction with Taq Polymerase to obtain fidelity of pfu with the speed of Taq Polymerase activity.
Real time PCR is also known as Quantitative Flourescence PCR(QF-PCR)/ qPCR. Real time PCR quantitatively measures the Amplification of DNA . SYBR Based Detection: Uses SYBR Green dye (dsDNA binding dye). TaqMan Based Detection: Usesa Florogenic Probe specificto Target Genetodetect
target as it accumulates during PCR.
Reverse Transcription PCR/ RT-PCR is used to detect RNA Expression. In this, sample RNA is converted to cDNA by enzyme Reverse Transcriptase.
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.
Quantitative Flourescent PCR (QF-PCR) use STRs to detect Aneuploidy. PCR is specific, simple, sensitive, flexible, rapid and relatively inexpensive technique
which amplifies DNA from low quantities quanti ties and can work on damaged DNA. DNA.
PCR has many applications such as Structural Analysis, DNA Typing, Disease
Detection, Cloning, Detection of Gene Expression, Mapping, Site Directed Mutagenesis and Sequencing. Ligase Chain Reaction (LCR) or Ligation Amplification Reaction (LAR) is a method for Amplification of DNA in which Probe is amplified. In LCR, enzyme used is thermostable DNA Polymerase and thermostable DNA Ligase (Taq DNA Polymerase and Taq DNA Ligase). Multiplex Ligation-Dependent Probe Amplification (MPLA) is a kind of multiplex PCR technique that can simultaneously analyze multiple Genomic regions. MLPA has small sample requirement. MLPA can detect copy number variations and can detect deletions and duplications of any size. Karyotyping is the best technique for Monosomy/Trisomy but can be done only during Metaphase of Chromosome. Karyotypingcannotbedonein anyphase ofcell cycle. It cannot DetectAmplifications, Micro-deletions and Complex Translocations. FISH (Fluorescent In-Situ Hybridization) is a rapid/fast technique which can be done in any phase of cellcycle. FISH can be done in morphologically intact cell/tissue/organ. FISH can detect Monosomy/Trisomy, Micro-deletions, Amplifications and
Complex Translocations. FISH tells Gene location on a Chromosome.
84 : Molecular
Restriction Restri ction Fragment L Length ength Polymorphism (RFLP) is a lengthy procedure and can detect only onl y single mutation and those mutation mutationss which which are affected by Pallindrome. P allindrome.
Probe is a DNA sequence, which is complementary to Gene of Interest or only a
small portion on Gene of Interest.
Probe
is labelled with a Radioactive material or a Fluorescent Dye. Hybridization/Blotting is used for visualization of sample DNA.
Technique Technique
SampleAnalyzed
Probe Probe
Gel Used Used
Southern Blot/ DNA-DNA Hybridization
DNA
DNA
Yes
Northern Blot/ Northern RNA-DNA Hybridization
RNA
DNA
Yes
Western Blot/ Immune Blot
Protein
Antibody
Yes
South Western Blot
Protein-DNA
DNA
No
Microarray
mRNA or cDNA
DNA
No
ELISA
Protein or Antibody
Specific Antibody for Protein to be detected
No
Single Gene Expression Analysis can be done by both Northern and Western Blotting. In Microarray/chip, small DNA fragments or Probes are immobilized on a solid support e.g. glass slide, in an ordered fashion. Microarray can detect Multiple Mutations, Multiple Gene Expression Analysis, Global Pattern of Gene Expression, SNPs, Genetic Transfer of Disease.
Microarray cannot detect Aneuploidy i.eMonosomy/Trisomy. Comparative Genomic Hybridization (CGH) or Chromosomal Microarray Analysis (CMA) is a cytogenetic method for analysis of copy differences between two genomes. If hybridization is done on Microarray plate (instead of using metaphase chromosomes) then is known as Microarray based CGH. CRISPR – Clustered Regularly Interspersed Short Pallindromic Repeats. Cas CRISPR CRISPR Associated Proteins. Pr oteins. Cas 9 an Endonuclease. CRISPR-Cas9 System is known as RNA Guided Nuclease system. CRISPR-Cas9 System is in Prokaryotes, to destroy Bacteriophage DNA. CRISPR and Cas Endonuclease acts together to degrade the Target DNA.
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 85
Conventional Karyotyping, FISH and CGH are cytogenetic techniques techni ques for Detection
of Mutations. Down Syndrome : 3 copies of Chromosome 21 Edwards Syndrome : 3 copies of Chromosome 18 PatauSyndrome : 3 copies of Chromosome 13
FISH, RT-PCR, QF-PCR, MPLA are the methods for the detection of Specific
Chromosomal Aneuploidy. Sperm Sorting (Microsort Method) – separate X-bearing (larger separate (larger in size & has more DNA) DNA) & Y-bearing sperms to be used for Artificial Insemination or In-Vitro Fertilization. Sanger’s Reagent is 1-Fluoro 2,4 Dinitro Benzene, used in DNA Sequencing. 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
RNA Interference/Gene Knock Down/Silencing Technique is a biological
phenomenon phenomeno by which which RNA molecule moleculess inhibit inhibit Translation. Translation. Gene KnocknDown - means Gene is present but its function is suppressed. Gene Knock Out - means Gene isdeleted. end of target mRNA mRNA to inhibit Gene Expression. Micro RNA binds to 3’ end Drosha workswithDGCR8 , alsoknownas PASHA. It cuts primary microRNA to
form pre-micro RNA. Dicer cut pre-microRNA to form dsRNA (around 20 Nucleotides in length). ss micro RNA combines with Proteins to form RISC (RNA-induced Silencing Complex). RISC can bind to the target mRNA and can just suppress it or degrade it by enzyme Slicer/Argonaute/Ago . 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.
CHAPTER MISCELLANEOUS MISCELLANEOUS
8 Ribosomes are organelles but they are Amembranous. Nucleus is rich in rRNA and it disappears during cell division. Mitochondria have their own mitochondrial DNA, RNA and Ribosomes which
synthesize around 10% of the Mitochondrial Proteins.
Lipofuscin/Residual Bodies are formed when indigestible material gets accumulated in lysosomes. lysosomes.
Pompe’s disease is the only Glycogen Storage Disease which is a Lysosomal Lysosomal Storage St orage Disease. Rest all Glycogen Glycogen Storage Disease Di sease affects Cytoplasm. Cytoplasm.
Most severe PBD (Peroxisome Biogenesis Disorders) is Zellweger Syndrome (Cerebro-Hepato-Renal Syndrome).
Micrososmes do not exist in a cell. During cell fractionation, Rough ER is disrupted to form Small Vesicles known asMicrososmes.
Strongest covalent bond is Peptide Bond/Amide Bond. Weakest bond – Vanderwaals Interactions. Selenium deficiency can lead to Hypothyroidism.
Three hormones involved in Homeostasis of Blood Calcium levels are Vitamin D,
Parathyroid Hormone & Calcitonin.
Keshan’s Disease occurs due to Selenium Deficiency. Acrodermatitiss enteropathic Acrodermatiti enteropathicaa - Disorder of ZincMetabolism.
Acrodermatitis enteropathica (moist erythematous plaques)
occurs urs when Concentration of Fluorine is > 20 ppm. Fluorosis occ Vitamins which can cause Dementia - Vitamin B1, B3 and B12.
88 : Miscellaneous Miscellaneous Nutritional causes of Cardiomyopathy are Deficiency of Thiamine, Selenium, Ca ++, Nutritional Mg++ and Excess of Iron.
Micronutrients in Glycolysis: Vitamin B1, B2, B3, Mg (for kinases). ++ ++ Minerals required in Energy - Mg , Zn , Chromium.
Vitamins those are necessary for Function are Thiamine, Pyridoxine Neurological and Cobalamine.
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.
Factors which increase Iron Absorption : Vitamin C, Amino Acids containing – SH SH group.
Factors which decrease Iron Absorption : Antacids, Alkalies, Phosphates, Phytates. +2 Fe is required for Iron Absorption. +3 Fe is required for Iron Transport & Storage. +2 Haem contains Fe . Factors which increase Calcium Absorption : Vitamin D, PTH (Parathyroid
Hormone), Lysine, Arginine.
Phytates decrease Calcium Absorption.
Vitamin K in its Coenzyme form is regenerated by enzyme Epoxide Reductase/ Vitamin K Reductase (required NADPH). Epoxide Reductase is inhibited by Oral Anti-Coagulants like Warfarin . Enzyme decreased in Lead Poisoning ALA Dehydratase. Enzyme increased in Lead Poisoning ALA Synthase. In Lead Poisoning, acid excreted in urine δ-Amino Levulinic Acid. ALA-Synthase I Deficiency is Lethal. ALA-Synthase II deficiency is X-linked Sideroblastic Anemia. ALA-Synthase II is Rate Limiting Enzyme of Haem Synthesis in Erythroid Tissues. Most common Porphyria is Porphyria Cutanea Tarda (PCT). Every 3rd Amino Acid in Collagen is Glycine. Every 7th Amino Acid in Elastin is Valine. Most of Proline & Lysine of Collagen is converted to Hydroxy-Proline & HydroxyLysine. 3 enzymes required for Collagen Formation are Prolyl Hydroxylase, Lysyl Hydroxylase and Lysyl Oxidase. Desmosine Cross Links are present in Elastin. Aldol Condensation (Intramolecular Cross Links) are present in Collagen. The most sensitive Acute Phase Reactant is CRP (C-Reactive Protein). Albumin is the most abundant Protein in Plasma. Albumin transports Most Steroids, T3, T4, Fat Soluble Hormones, Unconjugated Bilirubin, Calcium, Magnesium and manydrugs.
Normal Norm al A/G ratio is 1.2-1.6. Ceruloplasmin is a Cu containing enzyme, which also has Ferroxidase Activity.
Miscellaneous : 89 89
Ceruloplasmin transports Copper. Transferrin transports Iron. Transcortin transports Cortisol, Aldosterone & Progesterone . Haptoglobin transports Free Haemoglobin released from RBCs.
Hemopexin transports Free Heme released from Haemoglobin. Haemosiderin and Ferritin are storage forms of Iron. Haemosiderin has higher Iron content than Ferritin. Hepcidin regulates Iron transport in circulation. Afamin transports Vitamin E. Alpha- Feto Protein transports Long Chain Fatty Acids & Bilirubin . Transthyretin transports Thyroxine & Retinol Binding Protein . OFR with Highest Activity (most powerful) is OH (hydroxyl).
H2O2 is not a free radical but it can generate Free Radicals. Precursor of all ROS is
Superoxide Radical. MDA is not considered the Marker of Lipid Peroxidation.
derived Relaxation Factor) is also a Free Radical. Nitric Oxide– (Endothelium FOX Assay (Ferrous oxidation in Xylenol) is for the measurement of Free Radicals. Chain Breaking Anti-Oxidants are α-Tocopherol, β-Carotene and Superoxide
Dismutase (SOD).
Vitamins with Anti-Oxidant Activity: Vitamin A, C, E. Vitamin E is the most powerful Lipid Phase Antioxidant. Lipids are most susceptible for Free Radical Damage (known as Lipid Peroxidation). Enzymatic Antioxidants are Superoxide Dismutase, Catalase & Glutathione
Peroxidase.
Diabetic situation isalmost samelike Fasting or Starvation Increasedβ -oxidation
of Fatty Acids, Lipolysis, Proteolysis, Gluconeogenesis, Ketone Body Synthesis. In Diabetes: Snow Flake Cataractoccurs. Alcohol has Zero order kinetics. No Negative Feedback Control Negative Control for for Alcohol Metabolism. Ethanol & Fomepizole – used in Methanol Poisoning. Alcohol/Ethanol causes Fatty Liver due to increased NADH/NAD Ratio. Protein Precicipation can be done byHeat, Strong Mineral Acids, HeavyMetal Salts
and Alkaloidal Reagents (Trichloro Acetic Acid, Acid, Sulfosalicylc Sul fosalicylc Acid, Phosphotungstic Acid), Organic Solvents (ether) and Neutral Salts (Ammonium Sulfate).
Radio Immuno Assay (RIA) is used to assay Drugs, Vitamins, Hormones and to analyze Anti-DNA Antibodies in SLE (Systemic Lupus Erythematosus).
Most commonly used Method for HbA1C is Ion Exchange High Performance Liquid Chromatography (Ion Exchange HPLC). Best Method for HbA1C is Enzymatic Assay ( Horse-Radish Peroxidase enzyme used).
90 : Miscellaneous Miscellaneous Negative Negative charg chargee in Fibrinopeptide A & B is due to Acidic Amino Acids (Aspartate & Glutamate).
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.
Type IV Collagen (present in Basement Membrane of Glomerulus) is defective in Alport Syndrome. Type VII Collagen (present at the junction of Dermis & Epidermis) is defective in Epidermolysis Bullosa. 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 dieta ry Choline, Trimethylamine Oxide by Bacteria, Bacteria, Egg Egg Yolk, Liver, Fish. Trimethylamine is normallyoxidized in Liver by enzyme Trimethyl Amine Oxidase enzyme Oxida se to Trimethy Trimethyll Amine Oxide (odorless), (odorles s), 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).
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.
Calbindin is a group of several Calcium Binding Proteins. Subacute Combined Degeneration of Spinal Cord (known as Licht heim’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. Most abundant Pro form of Vitamin A is Beta-Carotene, which gets broken down to two molecules of Retinal.
The Secretory Vesicle are vesicles that mediate the Vesicular Transport of Substances - e.g. Hormones or Neurotransmitters from an organelle to specific sites by fusing with the t he Cell Membrane to release its content.
Secretory Vesicle
91 Miscellaneous : 91
energy ergy production production Substrate utilized for en FED
FASTING
STARVATION
Brain
Glucose
Glucose
Ketone bodies
Heart Liver
Fatty acids
Fatty acids
Ketone bodies
Glucose
Fatty acids
Amino acids
Glucose Muscle Adipose tissue Glucose Glucose RBC
Fatty acids
Fatty acids & K.B.
Fatty acids
Fatty Acids
Glucose
Glucose
Cognomen 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
Acids Specialized Products from Amino Acids Amino Acid
Metabolic Products
Aspartate
Arginine
Cysteine
Glutamate
N1 of Purine Purine N1, C4, C5, C5, C6 of Pyrimidine Pyrimidine Urea Synthesis
Nitric Oxide Oxide Agmatine Ornithine and Urea Creatine
Cystine Taurine Glutathione Beta mercapto ethanolamine N-Acetyl Glutamate N-Acetyl Glutamate Glutathione γ -Amino Butyric Acid
Miscellaneous 92 : Miscellaneous Amino Acid
Metabolic Products
N3 and N9 of of Purine Purine Pyrimidine N3 of Pyrimidine
Glutamine
Glycine
Histidine
Tryptophan
Tyrosine
Purine Heme Glutathione Creatinine FIGLU Histamine
Melanin Catecholamines (Epinephrine, Norepinephrine, Norepine phrine, Dopamin Dopamine) e) Thyroxine Serotonin Melatonin Niacin
Numbers Enzyme Code or Commision Numbers Enzyme
Enzyme Commision Number
Any Kinase
2
Thiokinase
6
Succinate Thiokinase
6
Any Phosphorylase
2
Glycogen Phosphorylase
2
Any Phosphatase
3
Glucose-6-Phosphatase
3
Any Mutase
5
Any Carboxylase
6
Any Dehydrogenase
1
Glycogen Synthase
2
Citrate Synthase
2
ATP Synthase
3
Nitric Oxide Synthase
1
Aconitase (TCA)
4
Fumarase (TCA)
4
Enolase (Glycolysis) PEPCK (Gluconeogenesis)
4 4
93 Miscellaneous : 93
Organelles Biochemical Markers of Various Organelles Nucleus
DNA, DNA, DNA Polymerase 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
Biochemistry Homophones in Biochemistry
D and L d and l
Racemic Mixture Racemase Enzyme
Dextrin Dextran
Dextrose
Lactose Lactulose
Lactate
Lactase
Lectin
Pectin Lignin
Insulin Inulin
Fructose1,6 Bisphosphate Fructose1,6Bisphosphatase Fructose1,6Bisphosphatase
Structural Isomers (Enantiomers Optical Isomers (Dextro-rotatory and Levorotatory) Equal d + l present(opticallyinactive) Enzyme which converts D and L (name Racemase is misnomer) Hydrolytic Product of Starch Branched, HMW Homopolysaccharide made up of α-Glucose (used as Plasma Plasma Volume Expander) Solution of Glucose Made up of Galactose + Glucose (Presentin Milk) Made up of Galactose + Fructose (usedas Laxative) Dead end of Glycolysis, Produced in Anaerobic Glycolysis Enzyme which breakdown Lactose (Disaccharide) Carbohydrate Binding Proteins, which play a role in Recognition and Attachment Polysaccharide, acting as a Soluble Dietary Fibre A complex organic polymer, which is the major Insoluble Dietary Fibre Hormone, secreted from β -Islet cells ofPancreas Pancrea s A Homopolysaccharide, made up of β-Fructose, also Ideal substance fo forr GFR
Involved in Glycolysis Involved in Gluconeogenesis
94 : Miscellaneous Miscellaneous
Fructose2,6 Bisphosphate
Fructose 2,6
Bisphosphatase Transferrin Transcortin Transthyretin Hemopexin Haemosiderin Hepcidin
Reciprocal regulator which increases the rate of Glycolysis and decrease the rate of Gluconeogenesis. Present in TIGAR (TP-53 Induced Glycolysis and Apoptosis Regulator). Transports Iron Transports Cortisol, Aldosterone & Progesterone Transports Thyroxine & Retinol Binding Protein Transports Free Heme released from Haemoglobin Are storage forms of Iron Regulates Iron transport in circulation
Peculiar Odours in Different Amino Acidurias Acidurias Inborn error of Metabolism
Urine Odour
Phenylketonuri a (PKU) Phenylketonuria Maple Syrup Urine Disease (MSUD)
Mousy/Musty 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
(MPS) Mucopolysaccharidosis (MPS) Disease Name
Enzyme Deficiency
Substrate Accumulated
I H-Hurler (Autosomal Recessive)
α-L-Idouronidase
*DS+#HS
I S-Scheie (Autosomal Recessive)
α-L-Idouronidase
*DS
II Hunter (X-linked Recessive)
Iduronate Sulfatase
*DS+#HS
VI Maroteaux Lamy (Autosomal Recessive)
Aryl Sulfatase B
*DS
*DS – Dermatan Sulfate, #HS – Heparan Sulfate
95 Miscellaneous : 95
Hyperlipoproteinemias Hyperlipoproteinemias Disease Name
Enzyme Deficiency
Substrate Accumulated
Type I/Familial Hyperchylomicronemia (Autosomal Recessive)
Lipoprotein Lipase or Apo C-II
Type II-α/Familial Hypercholesterolemia (Autosomal Dominant)
Absent or Defective LDL-Receptors or Apo B-100
Type II-β/Familial Hyperlipoproteinemia
Unknown defect
Type III/ III/ DysbetaLipoproteinemia/ Broad Beta Disease (Autosomal Recessive)
Apo E
Chylomicrons VLDL TGs (Triglycerides) Cholesterol LDL VLDL LDL TGs Cholesterol Chylomicron remnant VLDL remnant/IDL TG Cholesterol
Sphingolipidoses Sphingolipidoses Disease Name Name
Enzyme Deficiency
Substrate Accumulated
Neimann-Pick Disease Neimann-Pick Disease (Autosomal Recessive)
Sphingomyelinase
Sphingomyelin
Gaucher’s Disease (Autosomal Recessive)
β-Glucosidase/ Gluco-cerebrosidase
Glucosyl Ceramide
Krabbe’s Disease (Autosomal Recessive)
β-Galactosidase
Galactosyl Ceramide
Fabry’s Disease
α-Galactosidase
Ceramide Trihexoside
(X-linked Recessive) Farber’s Disease (Autosomal Recessive)
Ceraminidase
Ceramide
Tay Sach’s Disease (Autosomal Recessive)
Hexosaminidase A
GM2 Ganglioside
Sandhoff’s Disease (Autosomal Recessive)
Hexosaminidase A&B
GM2 Ganglioside
Metachromatic Leukodystrophy (Autosomal Recessive)
Aryl Sulfatase A
Cerebroside Sulfate
GM1 Gangliosidosis
β-Galactosidase
96 : Miscellaneous Miscellaneous
Glycogen Storage Diseases (GSD) (GSD) Disease Name
Enzyme Deficiency
Substrate Accumulated
Von-Gierke’s Disease Glucose-6-Phosphatase
Glucose-6-Phosphate
Pompe’s Disease Cori’s Disease
Acid Maltase Debranching Enzyme
Glycogen in Lysosomes Lysosomes Limit Dextrins
Anderson Disease
Branching Enzyme
Amylopecti Amylopectin n
Mc Ardle’s Disease
Muscle Phosphorylase
Muscle Glycogen
Her’s Disease
Liver Phosphorylase
Liver Glycogen
Urea Cycle Disorders Disorders Disease Name
Enzyme Deficiency
Substrate Accumulated
Hyperammonemia Type I
CPS-I (Carbamoyl Phosphate Synthetase-I)
NH3
Hyperammonemia Type II
OTC (Ornithine Transcarbamoylase)
NH3 OMP UMP Orotic Acid
Citrullinemia Type I
Argino-Succinate Synthetase
NH3 Citrulline
Arginosuccinic Aciduria
Argino-Succinate Lyase Lyase
NH3 Arginosuccinic Acid
Hyperargininemia Hyperarginine mia
Arginase
NH3 Arginine
Citrullinemia Type II
Citrin (Aspartate-
Citrulline
HHH Syndrome (Autosomal Recessive)
Glutamate Transporter) Ornithine transporter
Hyperammonemia Hyperornithinemia Homocitrullinuria
Miscellaneous : 97
Enzymes Rate Limiting Enzymes Pathway
Rate Limiting Enzymes
Regulators
Glycolysis
Phosphofructokinase-I
AMP , Fructose 2,6 Bisphosphate ATP , Citrate
TCA/Kreb’s Cycle/
3 Enzy Enzymes mes depending dependin g upon various conditions (Citrate Synthase, Isocitrate Dehydrogenase, α-Keto Glutarate Dehydrogenase)
ADP ATP , NADH NADH
Glycogenesis
Glycogen Synthase
Glucose-6-Phosphate , Insulin Epinephrine , Glucagon
Glycogenolysis
Glycogen Phosphorylase
Epinephrine , Glucagon , AMP
Citric Acid cycle
Glucose-6-Phosphate , Insulin , ATP
Gluconeogenesis
Pyruvate Carboxylase, PEPCK, Fructose 1,6 Bisphosphatase
Citrate AMP , Fructose 2,6 Bisphosphate
HMP
Glucose-6-Phosphate Dehydrogenase
NADP NADP NADPH NA DPH
Fatty Acid Oxidation Oxidation
Carnitine Acyl Transferase-I
Malonyl CoA
Fatty Acid Synthesis Synthesis
Acetyl CoA Carboxylase
Insulin , Citrate Glucagon Gluca gon , Palmitoyl Palmitoyl CoA
Ketone Body Synthesis Cholesterol Synthesis
HMG CoA Synthase
Glucagon , Insulin , Malonyl Malonyl CoA
HMG CoA Reductase
Insulin , Thyroxine Glucagon Gluca gon , Cholesterol, Cholesterol, Mevalonate , Statins , Bile Acids
PRPP Glutamyl Amido Transferase
AMP , Inosine
Purine Nucleotide Synthesis
Purine Catabolism/ Xanthine Oxidase Uric Acid Synthesis Synthesis
Monophophate(IMP) GMP -
,
Miscellaneous 98 : Miscellaneous Pathway
Rate Limiting Enzymes
Regulators
ATP , PRPP UTP
Carbamoyl Phosphate Synthetase-II (CPS-II)
Pyrimidine Synthesis
N-Acetyl Glutamate
Bile Acid Synthesis Synthesis 7-α Hydroxylase Carbamoyl Phosphate Urea Cycle
NAG NA G (N-acetyl (N-acetyl glutamate) glutamate)
Synthetase-I (CPS-I)
Porphyrin/Haem Synthesis
δ-Amino Levulinic Acid Synthase (ALA Synthase)
Haem
Table of Vitamins Vitamins Name
Vitamin B1/ Thiamine
Vitamin B2/ Riboflavin/ Warburg Yellow Enzyme
Vitamin B3/ Niacin/ Nicotinic Acid/
Active Form TPP (Thiamine Pyrophos phate)
FMN FAD
NAD+ + NADP
Function Oxidative decarboxylation reactions Transketolase Phosphorylation of chloride channel in brain
Electron transfer
Deficiency
(Rare) Riboflavinosis Dermatitis Angular Stomatitis Cheliosis
Electron transfer
Pellagra (Diarrhea, Dementia, Dermatitis,
Vitamin B6/ Pyridoxine/ Pyridoxamine/ Pyridoxal
RBC transketolase activity to be determined for biochemical assessment of deficiency
For detecting deficiency, RBC glutathione reductase is assessed Endogenously synthesized by bacterial flora
Tryptophan synthesizes it 60mg tryptophan
Death)
Nicotinamide
Vitamin B5/ Pantothenic Acid
Beri-Beri WernickeKorsakoff Syndrome
Special Features
Coenzyme A ACP
PLP (Pyridoxal Phosphate)
Acyl carrier TCA Haem synthesis
Transamination Simple Decarboxylation TransSulfuration
(Rare) Burning Feet Syndrome
(Rare) Neurological Manifestations Epileptic convulsions in infant
synthesizes of Niacin 1mg Contains β-Alanine in its structure Present in CoA and Acyl Carrier Protein (ACP)
Deficiency can be induced by Isoniazid
Miscellaneous : 99 99 Name
Active Form Biocytin (Biotin+ Lysine)
Vitamin B7/ Vitamin H/ Biotin
Vitamin B9/ Folic Acid
Vitamin B12/ Cobalamine Cobalamine
Tetrahydrofloic Acid (THF)
Methyl-co balamine Deoxyadenosyl
Function Carboxylation reactions Regulation of Cell Cycle
Carrier of one Carbon Unit Synthesis of Methionine, Purine, Thymidine Monophosphate
Cofactor for reactions:
Methionine synthase
cobalamine
Vitamin C/ Ascorbic Acid Acid
Ascorbic Acid
Deficiency
(Rare) Leiher’s Disease Disease Exfoliative Dermatitis
Megaloblastic Anemia tube defect Nueral
Hyperhomocysteinemia Pernicious Anemia
aminomutase Methylmalonyl CoA mutase
Anemia Dementia Spinal degeneration
Leucine
Antioxidant – – cofactor for hydroxylation reaction: Proline → HydroxyProline Lysine →
Special Features Consumption of Raw Egg White can induce Biotin Deficiency Synthesized by Intestinal Flora
Histidine load test/ FIGLU Excretion test done to find out Latent Folic Acid deficiency
Folate Trap due to Vitamin B12 deficiency In pernicious
anemia, B12or is given IM oral
Megaloblastic
Scurvy
Can cross placenta
Bitot’s spots spots blindness Night Growth retardation Xerophthalmia
Rickets (children) Osteomalacia (adults)
HydroxyRetinol Vitamin A/ Retinal Retinol/ Retinoic Retinal/ Retinoic Acid/ Acid β-Carotene
Vitamin D/ Cholecalciferol/ Ergocalciferol
1, 25- dihydroxy Cholecalciferol (Calcitriol)
Lysine
Reproduction Vision Growth and Differentiation Gene Expression
Calcium and Phosphate Absorption and Reabsorption
Excess Vitamin A is toxic, leads to fractures
Excess is toxic
100 : Miscellaneous Miscellaneous Name
Vitamin E / α -tocopherol -tocopherol
Vitamin K/ Menadione/ Menaquinone/ Phylloquinone
Active Form Any of the several tocopherol derivatives
Menadione Menaquinone Phylloquinone
Function Antioxidant Antiatherogenic Role
γ-Carboxylation of glutamate residue in clotting and other protein
Deficiency
(Rare)
Special Features
RBC fragility leads tohemolytic anemia Edema Nuerological problems Hemolytic anemia Retinopathy Dry skin and hair loss
In new borns Fat Malabsorption
High doses of Vitamin E depress Coagulability
Produced by GIT Flora
Minerals Table of Minerals Mineral
Major Functions
Sodium
Na+/K + ATPase pump Major Extracellular Cation Acid Base Balance Regulation of osmotic pressure transmission ion Nerve transmiss Maintainence of blood viscosity Electrolyte and Water Balance
Potassium
Chloride
Calcium
Na+/K + ATPase pump Major Intracellular Cation Electrolyte and Water Balance Regulation of pH of bodyfluids Maintainence of Neuromuscular excitability Cofactor for enzymes e.g. Pyruvate Kinase
Formation of HCl in stomach Regulation of osmotic pressure of extracellular fluids.
Calcification of bones andteeth Blood Clotting Decrease membrane Fluidity Capillary permeability and nerve excitabilty
Deficiency
Hyponatremia-
Excess Sodiumloss Excessivewater retention
Hypokalemia-
Muscular weakness Irregular heart hea rt beat Tachycardia Altered ECG pattern
Hypochloraemia-
Severe vomiting Addison’s Disease Respiratory Acidosis Metabolic Alkalosis
Rickets in Children Osteomalacia in Adults Osteoporosis in elderly
Miscellaneous : 101 101 Mineral
Major Functions
Phosphorus
Mineralization of bones and teeth Constituent of High energy phosphates like ATP
Magnessium
Iron
Copper
Selenium
Zinc
Constituent of Nucleic Acids Constituent of bones and teeth Cofactor for Kinases, Phosphorylases, Carboxylases Improves Glucose Tolerance Constituent of haem Component of oxido-reductase enzymes Electron transfer reactions Cofactor for Oxidases Fe absorption and its incorporation in the heme
Deficiency
Rickets Osteomalacia
Neuromuscular Neurom uscular weakness weakness
Second most prevalent nutritional deficiency Microcytic, hypochromic anaemia
Microcytic, hypochromic anaemia Menke’s Disease
Component of several enzymes or proteins involved in redox reactions Antioxidant Function Its action is complementary to Vitamin E Cofactor for Deiodinase Plays role r ole in Spermatogenesis Spermatogenesis Zinc fingers-Important constituent of regulatory proteins that control transcription Cofactor for Carbonic Anhydrase,
Keshan’s Disease (Endemic cardiomyopathy)
Poor Wound Healing Growth Retardation Infertility Hypogonadism
Acrodermatitis enteropathica
Alcohol Dehydrogenase, Dehydrogenase, Carboxy Lactate peptidase,, Alkaline peptidase Alkaline Phosphatase Phosphatase
Chromium
Molybdenum Molybdenum
Cobalt
Component of ‘Glucose Tolerance Factor’ Facilitates action of Insulin Regulation of Gene Expression
Cofactor for Xanthine oxidase,Sulfite Oxidase, Aldehyde oxidase, Lipoprotein Lipase
Constituent of Vitamin B12 Maturation of RBCs
Hyperglycemia Impaired Glucose Tolerance
Severe Neurological Abnormalities
Pernicious Anaemia
Miscellaneous 102 : Miscellaneous Mineral
Major Functions
Deficiency
Manganese
Required for Glycoprotein and Proteoglycan Synthesis Cofactor for mitochondrial SOD,
Pyruvate Carboxylase, Glutamine Synthesis Constituent of Thyroid hormones
Iodine
Flouride
Formation of Bones and teeth Provides resistance to tooth enamels to sustain acid attacks Potent inhibitor of activities of certain enzymes
Growth Retardation Bone Deformity Sterlity
Fatty Infiltration in Hepatocytes
Cretinism Goiter Myxedema
Dental caries Osteoporosis
ENERGETICS Number of ATPs in in Anaerobic Anaerobic Glycolysis Glycolysis = 2 ATPs/gluco ATPs/glucose. se. Number of ATPs in in Aerobic Glyco Glycolysis lysis = 7 ATPs/Gluc ATPs/Glucose. ose. ATPs formed by Substrate Level Phosphorylation in Glycolysis are 4 ATPs. ATPs formed by Oxidative Phosphorylation (ETC) in Glycolysis are 5 ATPs. Number of ATPs in in RBCs are never more than than 2 , but it can be less less than 2 if it is is RL (Rapaport Leubering) shunt. Link reaction gives 5 ATPs (as 2 NADH produced) from two Pyruvate. From one Glucose, two Pyruvate are obtained. One Glucose Glucose on complete breakdown or complete oxidation oxid ation = 32. One Glucose in a Cancerous Cell gives 2 ATPs (Warburg’s effect). One Acetyl CoA = 10 ATPs. One Palmitic Acid (16 C) =106. One Stearic Acid (18 C) = 120. 2 pyruvate to glucose 4 ATP & 2 GTP used. 2 llactate actate to glucose glucose 4 ATP & 2 GTP used. 2 alanine to glucose 4 ATP & 2 GTP used.
No ATPs used in:
HMP. Uronic acid pathway. Alpha oxidation. Oxidation of very long chain fattyacids. RL shunt. Synthesis of ketone body.
IMAGE BASED BASED
104 : Image Based Organelles of a cell cell
Different types of endocytosis endocytosis
Types of cell membrane transport systems s ystems
Image Based : 105 105 Barfoed’s Test
Test Molisch Test
Benedict’s Test
solution Blue solution
Green / yellow ppt ppt
Orange red ppt ppt
Brick-red ppt ppt
No sugar present present
Traces of sugar present present
Moderate amount sugar present present
Large amount sugar present present
Rothera’s test
Milky plasma plasma
Iodine test test
Seliwanoff’s test
106 : Image Based carbohydrates Osazones of various carbohydrates
Needle shaped glucosazone gluco sazone crystals as viewed under the microscope microscope
Galactosazone crystals as viewed under the microscope (rhombic plates) plates)
Powder puff/hedge hog shaped crystals of lactose as viewed under the microscope microscope
Sun flower shaped maltosazone crystals as viewed under the microscope microscope
cataract Snow flake cataract
Circumcorneal vascularization – Vitamin B2 deficiency deficiency
Oil drop cataract cataract
Bitot’s spots – Vitamin A deficiency
Image Based : 107 Kayser-fleischerringsin Wilson’s disease (copper deposited in eyes)
Menke’s disease
Self mutilation in Lysch Nyhann syndrome syndrome
Albinism Albinism
Gaucher’s cell
bodies Reilly bodies
Reilly bodies, also known as AlderAlder-Reilly Reilly bodies are found in many mucopolysaccharidosis 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. neutrophils.
108 : Image Based Enzymes Enzymes
inhibitor Competative inhibitor
Vmax same Km increases
Non-competative inhibitor inhibitor
Vmax decreases Km same
Michaelis menton graph (graph between velocity & substrate concentration)- concentration)- rectangular hyperbola hyperbola
Ion exchange chromatography chromatography
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