Biochemistry Revision E6.5.pdf

Biochemistry Revision 1 01 1 BIOCHEMISTRY REVISION 1 ----- Active space ----- Enzymes...

Biochemistry Revision 1 01 1 BIOCHEMISTRY REVISION 1 ----- Active space ----- Enzymes 00:00:14 Enzymes are specialized proteins → Act as biological catalyst. Exceptions : Ribozymes (RNA that acts as catalyst) like SnRNA, Peptidyl transferase, Ribonuclease P. Types of enzymes : Enzyme Simple enzyme Complex enzyme (Inactive) (Activator) (Whole enzyme/ Active) Only protein part Protein part Non-protein part (Apoenzyme) Coenzyme : Cofactor : Prosthetic : Low molecular wt Inorganic molecules. Covalently attached to organic molecules. Eg : Metals. apoenzyme. Eg : B complex vitamins. Examples of cofactor Enzymes requiring specific cofactor Zinc Carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase, ALP. Magnesium Kinase, phosphatases, enolase. Copper Tyrosinase (melanin synthesis), lysyl oxidase (covalent crosslinks of collagen), xanthine oxidase (purine catabolism). Molybdenum Xanthine oxidase, sulfite oxidase. Iron Heme : Cytochromes, nitric oxide synthase, tryptophan pyrrolase. Non heme : Succinate dehydrogenase, Aconitase. Classification of enzymes : 7 classes. Class no. Name of class Subclasses Remarks I Oxido reductase Dehydrogenase NAD+ (vit B3)/ FAD (vit B2)/ NADP+ (vit B3) are required. Oxidase Eg : MAO, cytochrome C oxidase. Oxygenase Eg : Hydroxylases (mono oxygenases) Peroxidase H2O2 → H2O. Eg : Glutathione peroxidase Catalase H2O2 → H2O. Present in peroxisome. Reductase Requires NADPH. Biochemistry Revision v1.0 Marrow 6.5 2023  2 01 Biochemistry ----- Active space ----- Class Class Name Definition Examples II Transferase Transfers functional Kinase, Any enzyme with ‘trans’. group III Hydrolase Breaks covalent bonds Digestive enzymes, Phosphatase, Arginase. by adding H2O IV Lyase Breaks covalent bond Any ‘Lyase’ named enzyme, Decarboxylase without adding H2O (Simple decarboxylation requires PLP). V Isomerase Produces isomers ‘Isomerase’ in its name, Racemace, Mutase. VI Ligase Require ATP Synthetase, Carboxylase (requires biotin). VII Translocase Ions channels/ pumps H+ pump, K+ channel. Mechanism of enzyme action 00:15:05 Lowering of activation energy : (Activation energy = Difference between the free energy of substrate & the transition state). The standard free energy change : ΔG0 ΔG0 = ΔGP - ΔGS (Gibb’s free energy : G) E : Enzyme; Enzymes lower the activation energy. S : Substrate; Enzymes do not alter ΔG (free energy change). P : Product. o Mechanisms to lower activation energy : Covalent catalysis. Acid base catalysis. Catalysis by strain Catalysis by proximity Theories behind the mechanism of action of enzymes : Conformational change Emil Fischer’s template theory/ Lock and key model Daniel Koshland’s theory/Induced fit theory Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 1 01 3 Factors affecting enzyme activity 00:22:02 ----- Active space ----- 1. Substrate concentration : Ist order Kinetics Zero order Kinetics V ∝ [S] Hyperbolic curve ↑ in substrate concentration → ↑ in rate of reaction → Till it reaches Vmax. Then no further ↑ in rate of reactions. Km : Michaelis constant Significance of Km : Denotes the affinity of enzymes towards the substrate. Higher the Km , lower is the affinity. Helps select the ideal substrate (with lower Km). Vmax S Vi : Initial velocity; Michaelis menten equation : vi = Vmax : Max. velocity. Km + S Line weaver burk plot/ Double reciprocal plot : y = ax + b → Equation of line. 2. Enzyme concentration : Enzyme concentration is directly proportional Velocity (V) to the velocity of reaction. V ∝ [E] → Straight line. Enzyme conc. [E] 3. Temperature : Vmax Bell shaped curve. Optimum temp. Optimum temperature in humans : 35°- 40°C. > 40°C : Sharp fall in rate of reaction d/t denaturation of enzymes. Vmax 4. pH of the medium : Bell shaped curve. Optimum pH Optimum pH : 5 to 9 (normal). Biochemistry Revision v1.0 Marrow 6.5 2023  4 01 Biochemistry ----- Active space ----- Enzyme inhibition 00:29:46 Competitive inhibition : Here, the inhibitor is a structural analogue of substrate and hence it competes with the substrate for binding to the active site. Examples : Most of the drugs. Malonate (poison) : Inhibits succinate dehydrogenase. Vmax1 = [S] [S] Vmax remains constant. X-intercept moves towards zero. Vmax1 = Vmax Y-intercept remain same. KmI > Km Plot is shaped like ‘X’. Non competitive inhibition : Without Inhibitor The inhibitor is not a structural analogue of substrate. It binds to a distinct site. E+P at a very negligible rate. With Inhibitor Eg : Most of the poisons. = Less products [S] [S] V < Vmax 1 max Vmax reduced → 1/Vmax (y intercept) increases. Km remains constant. Km constant → (-1)/Km (x intercept) unchanged. KmI = Km Plot is shaped like ‘v’. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 1 01 5 Uncompetitive inhibition : ----- Active space ----- The inhibitor has no affinity towards free enzyme but it binds to the enzyme-substrate (ES) complex. [S] [S] Vmax decreases. X-intercept moves away from zero. Km1 decreases. Y-intercept increases. Shape : Parallel. Summary : Uncompetitive Inhibition Noncompetitive Inhibition Competitive Inhibition Parallel V shaped X shaped Suicide inhibition/ mechanism based inactivation : The unreactive inhibitor, using the mechanism of enzyme, becomes reactive inhibitor & binds to the active site of enzyme irreversibly. Examples : Allopurinol inhibits Xanthine oxidase. Aspirin inhibits cyclooxygenase. Difluoro methyl ornithine inhibits ornithine decarboxylase. Enzyme regulation 00:41:44 Enzyme regulation Enzyme quantity Enzyme quality At the level At the level of gene of enzyme Enzyme degradation Covalent Allosteric Induction & Repression modification regulation Proteasomal Lysosomal By Ubiquitin Biochemistry Revision v1.0 Marrow 6.5 2023  6 01 Biochemistry ----- Active space ----- Allosteric regulation : Allosteric enzymes : Their activity at substrate binding (active) site is modulated by the presence of a modifier/effector. +ve modifier : Activator (Allosteric activation). -ve modifier: Inhibitor (Allosteric inhibition). Properties of allosteric enzymes : Multi-subunit enzyme (quaternary structure). Usually are rate limiting enzymes. Substrate saturation curve : Sigmoid curve. Follows a sigmoid kinetics d/t cooperative binding. Binding of one substrate favours binding of others to the same enzyme. Doesn’t follow Michaelis-Menten hyperbolic kinetics. Binding constant (S0.5 or K0.5 ) Allosteric activation and inhibition : Covalent modification/ hormonal regulation : Well fed state ( Post prandial) Fasting state (Post absorptive) High Insulin : Glucagon ratio. Low Insulin : Glucagon ratio. Enzymes in dephosphorylated state. Enzymes are in phosphorylated state. Glucagon → ↑ cAMP → Phosphorylation. Enzymes in glycolysis, glycogen synthesis, Enzymes in glucuneogenesis, glycogenolysis, fatty acid synthesis are active in fed state. lipolysis are active in fasting state. Kinetic constants : Vmax Catalytic constant or Kcat = Et Et : Total enzyme concentration (E + Es). Kcat Catalytic efficiency of enzyme = Km Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 2 02 7 BIOCHEMISTRY REVISION 2 ----- Active space ----- Introduction to carbohydrates 00:00:10 Carbohydrate : Aldose or keto derivative of polyhydroxyalcohol. Common monosaccharides : (Benedict’s test +ve). No. of carbon atoms Aldoses Ketoses Triose (simplest carbohydrates) Glyceraldehyde Dihydroxy acetone Tetrose Erythrose Erythrulose Pentose Ribose, Arabinose Ribulose, Xylose (Epimer of Ribose) Xylulose (Epimer of Ribulose) Hexose Glucose, Galactose, Mannose Fructose Common disaccharides : Reducing disaccharides : (Benedict’s test +ve). Non-reducing disaccharides : Name Monomer units Linkage Name Monomer units Linkage Maltose Glucose + Glucose α 1,4 Trehalose Glucose + Glucose α 1, 1 Isomaltose Glucose + Glucose α 1,6 Sucrose Glucose + Fructose α 1, β 2 Lactose Galactose + Glucose β 1, 4 Lactulose Galactose + Fructose α 1, β 4 Polysaccharides : 2 types. Homopolysaccharide. Eg : Glycogen, starch, cellulose. Heteropolysaccharide. Eg : GAG (mucopolysaccharides), pectin, agarose. Dietary fibres (aka non starch polysaccharide) : Complex carbohydrate, not digestible by humans, but fermented. Note : Lignin is neither digested, nor fermented. RDA is 40 g/2000 kcal. Soluble dietary fibre : In gums, mucilage, pectin. Insoluble (crude fibres) : In cellulose, hemicellulose, lignin. Glycosaminoglycans (GAG) 00:11:23 Unbranched polysaccharide containing repeating disaccharide units of Amino + Acid sugars. General properties of GAGs: Negatively charged Cushioning effect. Biochemistry Revision v1.0 Marrow 6.5 2023  8 02 Biochemistry ----- Active space ----- Special characteristics of GAG : GAG Special characteristics Keratan sulphate No uronic acid. Maintains corneal transparency. Hyaluronic acid No sulphate group. Not covalently linked to proteins. Facilitates cell migration during morphogenesis & wound repair. Compressibility of cartilage in weight bearing. Chondroitin sulphate Compressibility of cartilage in weight bearing. Most abundant GAG. Heparan sulphate Present in plasma membrane (act as receptors). Heparin Anticoagulant Dermatan sulphate Maintains the structure of sclera Mucopolysaccharidoses (MPS) : GAG is degraded in lysosomes. GAG Defect in GAG degradation → MPS (lysosomal storage disorder). All mucopolysaccharidoses are AR except Hunter’s disease. Core Protein Structure of GAG Most common MPS : Sanfilippo > Hunter > Hurler. Mucopolysacchridoses Enzyme defect Hurlers Disease (MPS I H) α L – iduronidase Scheie’ Disease (MPS I S) α L-iduronidase Hunter’s Disease (MPS II) L-iduronate sulfatase Sanfilippo disease (MPS III ) HS degradation Morquio disease (MPS IV) Galactosamine 6 sulphatase Maroteaux Lamy disease (MPS VI) N acetyl galactosamine sulfatase (Aryl sulfatase B) I Cell Disease (resembles MPS as it N acetyl glucosamine (GlcNAc) phosphotransferase affects GAG degradation) Features MPS I-H MPS I-S MPS II MPS III MPS IV MPS VI Mental deficiency + Absent + + Absent Absent Corneal clouding + + Absent Absent + + Visceromegaly + + + Absent Absent + Leucocyte (Reilly body) inclusions + + + + Absent + All MPS have coarse facial features, short stature, and dysostosis multiplex. Coarse facies Claw hand Corneal clouding Reily body inclusions Bullet shaped in leucocytes middle phalanx Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 2 02 9 Glucose transporters 00:28:31 ----- Active space ----- Classified as : Sodium dependent (SGLT) & sodium independent (Glut) Glucose Location Points to remember transporter SGLT 1 Intestine (Apical/ luminal side), Proximal Absorption of glucose. Unidirectional. tubules Na+ glucose symport. SGLT 2 Proximal tubules 2° active transport. Glut 1 Brain, kidney, placenta, colon, Widely located. retina, RBC High affinity for glucose (low km). Glut 2 Sinusoidal cells (liver), β cells (pancreas), Absorption of glucose. Intestine (serosal side), kidney Low affinity (high km). Glut 3 Neurons, placenta, kidney Very high affinity. Glut 4 Heart, skeletal muscle, adipose tissue Insulin dependent Glut. Glut 5 Testes, intestine Absorption of fructose. Glut 6 Spleen, leukocytes No transporter function (pseudogene). Glut 7 Liver, smooth ER Absorption of glucose. Glut 8 Blastocyst Glut 9 Intestine, kidney Urate transporter, Mutation : 1˚Gout. Applied aspects : Mutated SGLT2 → Lowers renal threshold → Renal glycosuria (Blood glucose normal, Urine Benedict’s test +ve). SGLT2 inhibitors (gliflozins) : Oral hypoglycemic agents, S/E : UTI. ORS : Glucose + sodium (SGLT 1 in intestine). Body in well fed state : High insulin : glucagon ratio Glut 2 in liver (high Km) Glucose → Glucose 6 phosphate Increased Glut 4 in heart, 1. Glycolysis → TCA cycle. muscle (↑glycolysis, ↑glycogen synthesis), 2. Excess glucose : Glycogen synthesis. adipose tissue (↑fatty acid synthesis). 3. HMP shunt : NADPH produced (used for fatty acid synthesis). Body in fasting state : ↓ Low insulin : glucagon ratio → Enzymes in phosphorylated state → Pathways activated : Glycogenolysis (in liver), gluconeogenesis Glycolysis/ EMP pathway 00:43:53 In all organs (site : cytoplasm). Both aerobic & anaerobically. Biochemistry Revision v1.0 Marrow 6.5 2023  10 02 Biochemistry ----- Active space ----- Preparatory phase : (Irreversible) Hexokinase/glucokinase Glucose Glucose 6 phosphate 1 ATP 1 ADP (reversible) Phospho hexose isomerase Fructose 6 phosphate 1 ATP Phospho fructokinase 1 ATP used = 2 1 ADP Fructose 1,6 bisphosphate Aldolase (reversible) Dihydroxy acetonephosphate (DHAP) Glyceraldehyde 3 phosphate (G3P) Phosphotriose isomerase Pay off phase : Glyceraldehyde3 phosphate Gly 3 Po4 dehydrogenase Pi ETC NAD + 1 NADH = 2.5 ATP x 2 Reversible step Inorganic PO4 added NADH & H+ = 5 ATP 1,3 Bisphosphoglycerate (1,3 BPG) 1,3 BPG Kinase ADP (Substrate level phosphorylation) (only reversible kinase) ATP x 2 = 2ATP 3 phosphoglycerate 3 phosphoglycerate mutase 2 phosphoglycerate Enolase Mg2+/Mn2+ H20 Phosphoenol pyruvate (PEP) Pyruvate kinase ADP (Substrate level phosphorylation) (Irreversible step; Regulatory step) ATP x 2 = 2ATP Pyruvate Anaerobic glycolysis : Occurs in the RBCs where there are no mitochondria or lack of oxygen. No net generation of NADH. Glucose Lactate 2 Gly 3 Po4 NAD+ Lactate NADH dehydrogenase 1,3 BPG Pyruvate Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 2 02 11 Important enzymes : ----- Active space ----- Enzyme induced by insulin : Glucokinase. Enzyme that regulate glucose level following a meal : Glucokinase. Rate limiting step/Commited step of Glycolysis is PFK-1. Inorganic Phosphate is added in G3P Dehydrogenase. Irreversible steps of Glycolysis : All Kinases except 1,3 B P Glycerate Kinase. Substrate level Phosphorylation : 1,3 BP Glycerate Kinase & Pyruvate Kinase. Energetics : Pathway ATPs generated ATPs consumed Net ATPs Anaerobic Glycolysis 4 2 2 Aerobic Glycolysis 9 2 7 1 mol of glucose in aerobic condition (aerobic glycolysis = 7 ATP, 32 2 x Pyruvate →Acetyl CoA = 2 x NADH = 2 X 2.5 ATP = 5 ATP 2 x TCA cycle = 2 x 10 ATP = 20 ATP) Pyruvate dehydrogenase 00:54:13 Site : Mitochondria. Multienzyme complex : 3 enzymes 5 coenzymes E1 : Pyruvate dehydrogenase Thiamine Pyrophosphate (vit B1). E2 : Dihydrolipoyl transacetylase Lipomide. E3 : Dihydrolipomide dehydrogenase FAD (vit B2), NAD (vit B3), CoA (vit B5). Net reaction (oxidative decarboxylation) : Significance of pyruvate dehydrogenase : Link reaction (Links glycolysis and TCA) Enters TCA Excess PDH Carbohydrates Glucose Pyruvate Acetyl CoA Fatty acids Irreversible + Glycerol No enzyme TAG (Fats) Fat (Triacylglycerol) can’t be converted to Glucose except Glycerol & PropionylCoA. Clinical significance : (Leigh syndrome) Metabolic defect (PDH Complex : E1 MC affected) → No production of Acetyl CoA → Increased pyruvate → Converts to lactate → Lactic acidosis. Biochemistry Revision v1.0 Marrow 6.5 2023  12 03 Biochemistry ----- Active space ----- BIOCHEMISTRY REVISION 3 Glycogen metabolism 00:00:20 Glycogen present in : Liver (source of blood glucose during fasting). Skeletal muscle cells (energy during exercise). Glycogen linkage : Linear side (α 1, 4 glycosidic linkage). Branching point (α 1, 6 linkage). Glycogen synthesis : 1. Synthesis of UDP glucose. 2. Action of glycogen synthase → Glycogenolysis : In early fasting state (4-16 hours without food). 1. Action of Glycogen phosphorylase : Rate limiting enzyme. Coenzyme : PLP (Vitamin B6). Creates polymers of glucose with short branches (Limit dextrin) 2. Debranching enzyme (Bifunctional with tandem action). 3. Conversion of glucose-1-PO4 to glucose Liver : Mediated by G-6-phosphatase (in SER). Muscle : PGM G-1-p G-6-P Pyruvate Lactate Anaerobic glycolysis 3 ATP (no hexokinase step) Factor favouring glycogen synthesis Factors favouring glycogenolysis Insulin Glucagon & epinephrine Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 3 03 13 Glycogen Storage Disorders (GSD) 00:13:43 ----- Active space ----- Types Enzyme defect Clinical features Doll like facies, thin extremeties. Type 1/ Massive hepatomegaly. Glucose 6 Von Gierke’s Biochemical hallmarks : Phosphatase. (Liver GSD). Hypoglycemia, HyperuricemiaHyperlipidemia Lactic acidosis, Ketosis. Type 11 / Pompes Acid α 1-6 HCM & cardiomegaly. (Muscle GSD) : glucosidase/ Acid Floppy infant (extreme hypotonia). lysosomal defect. maltase. Child die by 2 years (cardiac failure). Type 111 / Cori’s/ No lactic acidosis, No hyperuricemia. Limit dextrinosis. Debranching enzyme. Liver cirrhosis +. (Liver GSD) : Liver enzymes (AST & ALT). Abnormal glycogen. Type IV/Anderson No lactic acidosis, No hyperuricemia. Branching enzyme. /Amylo-pectinosis. Liver failure (fatal) : Dies by 5 years Liver enzymes (AST & ALT). Exercise intolerance Type V /McArdle’s Muscle glycogen 2nd wind phenomenon seen. (Muscle GSD) phosphorylase. S.lactate after exercise. Type VI / Her’s Hepatic glycogen No severe hypoglycemia. (liver GSD) phosphorylase. Exercise intolerance, Hemolysis. Type VII / Tarui’s Muscle & erythrocyte No 2nd wind phenomenon. (Muscle GSD) phospho-fructokinase. S.lactate after exercise Fanconi Bickel GLUT 2 syndrome (low affinity) Type 0 Glycogen synthase No glycogen deposition ; No Hepatomegaly Liver GSD : Type I, III, VI, O. Muscle GSD : Type V, VII, O, II. Von Gierke’s Pompe’s Regulation of glycogen metabolism 00:19:07 Types : 1. Covalent modification. In fed state : Insulin activates phosphatase → activates glycogen synthase & inactivates glycogen phosphorylase. Biochemistry Revision v1.0 Marrow 6.5 2023  14 03 Biochemistry ----- Active space ----- In fasting state : Glucagon activates cAMP dependent protein kinase which in turn activates Glycogen phosphorylase. Note : Calcium from SER of contracting muscle activates glycogen phosphorylase via Ca-calmodulin complex & causes glycogenolysis. 2. Allosteric modification : One liners : Liver glycogen is depleted after 12- 18 hours of fasting. Common enzyme for glycogenolysis & glycogen synthesis : Phosphoglucomutase. First product of Glycogenolysis : Glucose 1 phosphate. Glycogenolysis enzyme absent in the muscle : Glucose 6 phosphatase. Rate limiting enzyme of glycogen synthesis : Glycogen synthase. Glycogen synthase is active in dephosphorylated state. Glycogen phosphorylase is active in phosphorylated state. Glucagon & Epinephrine acts via cAMP dependent protein kinase A. c AMP independent Ca- Calmodulin dependent activation of glycogenolysis is present. 5’AMP is an allosteric activator of muscle phosphorylase. HMP pathway 00:39:18 aka pentose phosphate pathway/Dicken Horecker pathway. Organelle : Cytoplasm. No ATP generation. Rate limiting step : Glucose 6 phosphate dehydrogenase. HMP phase 1 (oxidative phase) HMP phase 2 (non oxidative phase) Irreversible Reversible Generate NADPH Generate pentoses → DNA synthesis G6PD (rate limiting enzyme) Produces glyceraldehyde-3-PO4→ Glycolysis→ Acetyl CoA Function of NADPH : 1. Reductive biosynthesis of fatty acids & cholesterol : Occurs in liver, adipose tissue, adrenal cortex, gonads. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 3 03 15 2. Free radical scavenging (H2O2 → H2O) : ----- Active space ----- Occurs in RBC (membrane integrity) & lens (transparency). 3. Respiratory burst : Phagocytosis by WBC. NADPH oxidase require NADPH. Disorder associated : Chronic granulomatous disease. 4. Nitric oxide synthase : Require NADPH. 5. Cyt P450 (Phase 1 detoxification reaction) : Require NADPH. Other sources of NADPH : Isocitrate dehydrogenase (cytoplasmic). Malic enzyme( NADP Malate Dehydrogenase). G6PD deficiency (XLR) : M/c enzyme deficiency in human beings (2nd m/c : Pyruvate kinase). Deficiency of NADPH. Precipitated by sulfa drugs, fava beans. Clinical features : 1. Hemolysis (jaundice + anemia). 2. Methemoglobinemia. Erythrocyte transketolase is an indicator of thiamine status. Gluconeogenesis 00:48:16 In late fasting state (16-48 hours without food). Organelle : Cytoplasm + mitochondria. Non-carbohydrate substrates converted to glucose : Lactate : Glucose lactate cycle (corI’s cycle). Glucogenic amino acids (Alanine) : Glucose alanine cycle (Cahill cycle). Glycerol. Propionyl CoA. Acetyl CoA is never a substrate. It is allosteric activator of pyruvate carboxylase. Key enzymes : Gluconeogenesis Glycolysis (irreversible steps) Pyruvate carboxylase. Pyruvate kinase PEP carboxykinase. Fructose 1,6 Bisphosphatase Phosphofructokinase Glucose 6 phosphatase Hexokinase Malate aspartate shuttle : Transport of oxaloacetate from mitochondria to cytoplasm. Biochemistry Revision v1.0 Marrow 6.5 2023  16 03 Biochemistry ----- Active space ----- Reciprocal regulation of glycolysis & gluconeogenesis : Enzymes Activator Inhibitor Phospho 5’ AMP. Citrate. Fructokinase-1 Fructose 6 Phosphate. ATP. Fructose 2,6 Bisphosphate. Low pH. Pyruvate Kinase Fructose 1,6 Bisphosphate. Pyruvate CoA , NAD+ , ADP ATP. Dehydrogenase Pyruvate. Acetyl CoA. NADH. Pyruvate Carboxylase Acetyl CoA. ADP. Uronic acid pathway/synthesis of ascorbic acid : Humans cannot synthesis vitamin C (d/t absence of L Gulanolactone oxidase). Polyol/sorbitol pathway : Responsible for development of cataract in DM. Galactose metabolism 01:01:01 Metabolised exclusively in liver. Galactose tolerance test : Liver function test. Types of galactosemia : 1. Classic galactosemia : Galactose 1 PO4 uridyl transferase defect. 2. Non classic galactosemia : a. Galactokinase defect : Oil drop cataract. b. UDP hexose epimerase defect. Fructose metabolism 01:07:18 Fructokinase Fructose Fructose 1 Phosphate Aldolase B Glyceraldehyde Dihydro acetone phosphate Glyceraldehyde 3 Phosphate Enter glycolysis Note : Phosphofructokinase (rate limiting step of glycolysis) is not involved in fructose metabolism. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 3 03 17 Metabolic disorder Enzyme Defect Features ----- Active space ----- Hereditary Fructose Aldolase B Resembles Intolerance (HFI) galactosemia. Essential Fructosuria Fructokinase Benign condition Essential pentosuria Xylulose reductase/Xylitol Benign condition. dehydrogenase Bial’s test +ve. Galactosemia HFI Age of onset First 2 weeks. ~ 6 months. Trigger Breast milk Weaning food Jaundice, seizures, lethargy. Resemble Clinical Failure to thrive , Hypoglycemia. galactosemia. features Oil drop cataract (Galactitol). But no cataract. Prone for E coli sepsis. Accumulating Galactose 1 Phosphate. Fructose 1 Phosphate substance (toxic to liver). Specific test (galactose, fructose) : Benedict test +ve for reducing sugars. Investigations Glucose oxidase test -ve for glucose. Direct enzyme assay. Treatment Non lactose diet. Withdraw breast milk. Fructose free diet. TCA cycle 01:12:23 Biochemistry Revision v1.0 Marrow 6.5 2023  18 03 Biochemistry ----- Active space ----- Inhibitors of TCA cycle : 1. Flouroacetate inhibits aconitase. 2. Arsenate inhibits alpha keto glutarate dehydrogenase. 3. Malonate inhibits succinate dehydrogenase. Truly an amphibolic pathway. Final common oxidative pathway of lipids, carbohydrates & Proteins. Acetyl CoA is completely oxidised. Unidirectional steps : Citrate synthase & α keto glutarate dehydrogenase. 3 NADH and 1 FADH2 are produced. Total No of ATPs generated by one turn of TCA cycle : 10 ATPs. Succinate dehydrogenase is linked to complex II of ETC. FADH2 is produced by Succinate dehydrogenase. Major anaplerotic (filling up) reaction in TCA : Pyruvate carboxylase. Vitamins in TCA cycle : 1. Pantothenic acid : In CoA. 2. Thiamine : In α KGOH. 3. Riboflavin : In FAD. 4. Niacin : In NAD. Oxidative phosphorylation 01:19:10 Oxidation coupled with phosphorylation by means of H+ gradient. Every reducing equivalent entering ETC is converted to ATP. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 3 03 19 Complexes of ETC Inhibitors ----- Active space ----- NADH Q oxidoreductase. Rotenone, Complex 1 PUmps 4 H+ into intermembrane space. Amobarbital, Piercididin A. Complex Succinate Q oxidoreductase. Malonate, II Does not pump H. + Carboxin, TTFA. Complex Q cyt c oxidoreductase/Cyt b -cyt C1 complex. Antimycin A, III PUmps 4 H+ into intermembrane space. BAL (Dimercaprol) Cyt C oxidase. CO, Cyanide, H2S, Complex Final electron acceptor. Sodium azide. IV PUmps 2 H+ into intermembrane space. Point of phosphorylation. Atractyloside, Complex Has 2 subunits : Oligomycin, V (ATP 1. F0 : Proton channel. Venturicidin. synthase 2. F1 : Various subcomplex (Gamma : Rotatory complex) channel, Beta : Convert ADP to ATP). Uncouplers of oxidative phosphorylation : Uncouplers of oxidative phosphorylation Chemical 2,4 Dinitrophenol ; Dinitrocresol ; Aspirin in high dose. uncouplers FCCP (Fluoro Carbonyl Cyanide Phenyl hydrazine). Physiological Thermogenin (uncoupling Protein I) , Thyroxine. uncouplers Long chain fatty acid. Ionophores Valinomycin & Gramicidin. High energy compounds : Compounds which yield energy on hydrolysis (atleast 7 KCal/mol). Examples : ATP, 1,3 Bisphosphoglycerate, Acetyl CoA, succinyl CoA, Phosphoenolpyruvate, Phospho creatine, Phospho arginine. Respiratory quotient (RQ) : Ratio of CO2 produced/O2 consumed. Substrate RQ Substrate RQ Carbohydrates 1.00 Mixed diet 0.80-0.85 Protein 0.81 Exclusive carbohydrate diet 1.00 Fat 0.71 Excess carbohydrate diet ≤ 1.00 Alcohol 0.66 Biochemistry Revision v1.0 Marrow 6.5 2023  20 04 Biochemistry ----- Active space ----- BIOCHEMISTRY REVISION 4 Lipids 00:00:17 These are heterogenous group of compounds insoluble in polar solvents but soluble in nonpolar solvents like chloroform or ether. Essential Semi-essential Omega 3 fatty acids Omega 6 fatty fatty acids fatty acids acids Linoleic acid Arachdonic acid. A : Alpha linolenic acid. G : GLA T : Timnodenic acid. L : Linoleic acid. Alpha- Gamma Linolenic C : Cervonic acid aka A : Arachidonic acid. linolenic acid acid (GLA). Docosahexaenoic acid (DHA). Docosa hexaenoic acid or cervonic acid : Low DHA associated with retinitis pigmentosa. Function : Development of foetal brain and retina. Trans fatty acid : Partially hydrogenated edible oil (margarine or cake butter). Most Unsaturated fatty acids exist in cis form, on heating Trans form. Present in bakery items, fried foods and processed food. Deleterious effect are : a. TAG , LDL, HDL. b. Cardiovascular risk. c. Inflammation. d. Insulin resistance. Omega 3 fatty acids : Cardiovascular risk. Replaces arachidonic acid (source of inflammatory markers) in platelets. Thromboxanes and tendency of platelet aggregation. inflammation. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 4 04 21 Richest sources of fatty acids : ----- Active space ----- Fatty acid Richest source SFA (Saturated Fatty Acid) Coconut oil PUFA (Poly UFA) Safflower > Sunflower. Least : Coconut oil. Linoleic acid Safflower oil. Alpha linolenic acid Flax seed oil MUFA Mustard/Rape seed oil. Trans fat Vanaspati/Dalda Phospholipids 00:09:05 Two types : 1. Glycerophospholipids 2. Sphingophospholipid 1. Glycerophospholipids : Glycerol + 2 Fatty acids(FA) + Phosphate + Nitrogenous base. Diacyl glycerol(DAG) Glycerophospholipid Constituents Uses Phosphatidic Acid (PA) DAG + Cell membrane Phosphate Lecithin/phosphatidyl PA + Choline Most abundant phospholipid in cell mem- choline brane. Lung surfactant. Store house of choline. Cephalin PA + Ethanol- Cell membrane. amine Cardiolipin PA + Glycerol Only antigenic phospholipid. (diphosphatidyl + PA Present in inner mitochondrial membrane. Glycerol) False +ve result in test for syphilis (VDRL). Associated with Barth syndrome/Cardi- oskeletal myopathy (mitochondrial disorder). Phosphatidyl serine PA + serine Apoptosis. Phosphatidyl inositol PA + Inositol Second messenger. 2. Sphingophospholipid : Sphingosine + FA + Phosphate+ Nitrogenous base. Ceramide Biochemistry Revision v1.0 Marrow 6.5 2023  22 04 Biochemistry ----- Active space ----- Sphingophospholipid Constituents Uses Sphingomyelin Ceramide + PO43- + Choline. Myelin sheath White matter of brain. Note : L/S Ratio (Lecithin/Sphingosine) : Indicator of lung maturity. In mature lung : More lecithin present. Glycolipids 00:15:15 Not a phospholipid. Ceramide + Carbohydrate. Glycosphingolipid Constituents Uses Cerebroside Ceramide a. Glucocerebroside : Extraneural tissue. + b. Galactocerebroside : Neural tissue. Monosaccharide Globoside Ceramide + Oligosaccharide Ganglioside Ceramide + Oligosaccharide + N-acetyl Neuraminic acid. (GM1, GM2, GM3) Sphingolipidoses : Defect in sphingolipids degradation (takes place in lysosomes). Disease Enzyme defect GM 1 Gangliosidoses ß Galactosidase. Tay Sach’s disease ß Hexosaminidase A. (GM 2 Gangliosidoses) Sandhoff’s disease ß Hexosaminidase A & B. (GM 2 Gangliosidoses) Krabbe’s disease ß Galactocerebrosidase/ ß Galactosidase Niemann Pick Type 1 Sphingomyelinase Gaucher’s disease ß Glucocerebrosidase/ß Glucosidase Metachromatic Aryl Sulfatase A leukodystrophy Farber’s disease Ceramidase Wolman’s Disease(LSD) (not an SPL) Acid Lipase Fabry’s disease α-Galactosidase In the table above, LSD-Lysosomal storage disorder, SPL-Sphingolipidoses. Gaucher’s Disease : M/C lysosomal storage disorder. No mental retardation (MR), no cherry red spot. Bleeding tendency, pain and pathological # of long bones. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 4 04 23 ----- Active space ----- Crumpled tissue paper Visceromegaly Erelenmeyer flask appearance on bone marrow deformity Fabry’s disease : XLR, No MR, hypohydrosis, Fabry’s crisis (pain in proximal joints). Angiokeratoma Maltese cross Whorled appearance in lens; Lenticular (urine microscopy) opacity Krabbe’s disease : Severe neurological deficit. No visceromegaly. Multinucleated globoid cell inclusion bodies in white matter of brain. Opisthotonus posture Wolman’s disease/cholesterol ester storage disorder : Not a sphingolipidoses, type of LSD. Deficiency : Acid lipase. C/F : Watery green diarrhoea. Pathognomic feature : Calcification of adrenals. Important points for sphingolipidoses : All are autosomal recessive except Fabry’s disease (X linked recessive). No cherry red spot on the macula : Fabry’s disease, Gaucher’s disease. No mental retardation : Fabry’s disease, Gaucher’s type I. Angiokeratoma : Fabry’s disease, GM 1 gangliosidoses. Resembles rheumatoid arthritis : Farber’s disease. No visceromegaly : Metachromatic leukodystrophy, GM 2 gangliosidoses, Krabbe’s disease. Zebra body inclusion : Niemann Pick’s disease. Biochemistry Revision v1.0 Marrow 6.5 2023  24 04 Biochemistry ----- Active space ----- Metabolism of lipids 00:27:22 Well fed state Fasting state High insulin/glucagon ratio. Low insulin/glucagon ratio. Lipogenesis Lipolysis Glycogenesis Gluconeogenesis TAG synthesis, cholesterol synthesis. Ketone bodies synthesis, fatty acid oxidation. Lipolysis : Acyl CoA Acyl CoA Acyl CoA TAG HSL DAG HSL 2,MAG MAG esterase Glycerol Hormone Sensitive Lipase (HSL) Stimulated by Glucagon, glucocorticoids, epinephrine, TSH, thyroxine, ACTH, MSH Inhibited by Insulin, nicotinic acid, PGE 1 Fatty acid oxidation : Stages of fasting Source of glucose Early fasting Glycogenolysis (4-16 hrs) Fasting Gluconeogenesis (16-48 hrs) Prolonged fasting (starvation) Lipolysis (2-5 days) Prolonged starvation Muscle proteolysis (> 5 days) Note : Glycerol released during lipolysis is a substrate for gluconeogenesis. FA oxidation is a source of ATP for gluconeogenesis. FA oxidation ketone body synthesis. Beta-oxidation of fatty acids : Site : Liver, adipose tissue, skeletal muscle. Organelle : Mitochondria. Steps of FA oxidation : (1). Preparatory. (2) Beta-oxidation. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 4 04 25 Preparatory : ----- Active space ----- a. Activation of FA : Occurs in cytoplasm. Acyl CoA Synthetase/Thiokinase FA Acyl CoA 1 ATP 1 AMP Uses : 2-PO43- b. FA transport : Cytoplasm → Mitochondria. Gateway of oxidation : CPT-1 (rate limiting enzyme). Malonyl CoA inhibits CPT-1. Note : FA < 14 C do not require Carnitine. Beta-oxidation : Energetics of beta oxidation : Acyl CoA No. Of acetyl CoA = No of C atoms FAD 2 FADH2 Acyl CoA Dehydrogenase 1 acetyl CoA = 10 ATP. α β Unsaturated Acyl CoA If number of Acetyl CoA = n Then no. of cycles of β-oxidation = n - 1 Hydratase + H20 1 cycle of β oxidation : (1 NADH + 1 FADH2) ATPs Hydroxy Acyl CoA (2.5 + 1.5) = 4 ATPs NAD+ Hydroxy Acyl CoA DH For palmitic acid (C16) : NADH+ H+ KetoAcyl CoA No of acetyl CoA = 16/2 = 8 → 80 ATP. No of cycles = 8-1 = 7 → 7 x 4 = 28 ATP. Acyl CoA Acetyl CoA Total ATP generated = 80 + 28 = 108 ATP. 2 ATP consumed by thiokinase → 106 ATP Disorders of fatty acid oxidation : (net ATP produced). 1. Jamaican vomiting sickness : D/t consumption of unripe Ackee fruit. C/F : Sudden onset of vomiting, fatal condition. Toxin : Hypoglycin (inhibits acyl CoA dehydrogenase) ß oxidation, Gluconeogensis & Ketone body synthesis. 2. MCAD deficiency (Medium Chain Acyl CoA Dehydrogenase deficiency) : Medium chain fatty acid oxidation is affected. Leads to SIDS (Sudden Infant Death Syndrome). Fatal condition. /no ß oxidation alternate pathway Omega (w) oxidation dicarboxylic acid. Diagnosis : Hypoglycemia, No ketone bodies. Treatment : Frequent meals with low fat and high carbohydrates. Biochemistry Revision v1.0 Marrow 6.5 2023  26 04 Biochemistry ----- Active space ----- Summary of oxidation of fatty acids : Fatty acid oxidation Site Important points Beta oxidation : Saturated fatty acid Mitochondria Unsaturated fatty acid Mitochondria Acyl CoA DH step is bypassed. 1.5 ATPS less for each double bond. Odd chain fatty acid Mitochondria (Acetyl CoA) X n + Propionyl CoA (glucogenic) Alpha oxidation Peroxisome & No ATPs (minor oxidation) Endoplasmic reticulum Omega oxidation Micrososmes (SER) No ATP Dicarboxylic acid produced. Zellweger syndrome/cerebrohepatorenal syndrome : CF : Frontal bossing, epicanthal folds, hypertelorism, mongoloid facies. Ophthalmic examination : Bruschfield spots in the iris. Defect : Mutation in PTS (Peroxisomal Targeting Sequence) → Defect in oxidation of VLCFA & alpha oxidation. Blood picture : S.VLCFA (very long chain FA). S. Phytanic acid. Peroxisomes are /absent. Refsum’s disease : Defect : Phytanoyl CoA hydroxylase/oxidase enzyme (alpha oxidation). C/F : Pigmentary retinopathy. Peripheral neuropathy. Icthyosis. Cardiac arrhythmia. Diagnosis : S. Phytanic Acid. Treatment : Avoid phytanic acid (green leafy vegetables, dairy products). Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 4 04 27 Ketone body synthesis : ----- Active space ----- Site : Exclusively in liver mitochondria. Note : Ketone body is not utilised in liver (thiophorase absent) and RBC (mitochondria absent). Acyl CoA β oxidation β-OH Butyrate (Acetyl CoA) X N Acetoacetyl CoA Mitochondrial HMG CoA Acetoacetate Synthase -RLE (rate limiting) Thiophorase HMG CoA HMG CoA Lyase Acetoacetyl CoA Acetoacetate (1˚ Ketone body) Acetyl CoA Acetyl CoA Dehydrogenase CO2 Acetone β-OH Butyrate RLE : Rate Limiting Enzyme. Acetoacetate & β-OH-butyrate are used in extrahepatic tissue. Acetone is excreted by lungs. Fatty acid synthesis : Organelle : Extramitochondrial (cytosol). Starting material : Acetyl CoA. Transporter of acetyl CoA : Citrate (tricarboxylic acid transporter). Enzyme that releases acetyl CoA : ATP citrate lyase. Acetyl CoA Carboxylase (RLE) Acetyl CoA(2C) Malonyl CoA(3C) ATP ADP Biotin CO2 Fatty acid synthase complex : 1. Condensing unit : Has 2 enzymes. 2. Reducing unit : Has 3 enzymes, NADPH required. 3. Releasing unit (thioesterase). 4. Acyl carrier protein (has vitamin B5). Biochemistry Revision v1.0 Marrow 6.5 2023  28 04 Biochemistry ----- Active space ----- Cholesterol synthesis : Exclusive animal sterol. Not a metabolic fuel. Organ : Liver, adrenal cortex, adipose tissue, gonads. Organelle : Cytoplasm, SER. Substrate : Acetyl CoA. Note : Statins inhibit HMG CoA reductase competetively. Vitamin D. Acetyl CoA Cholesterol Steroid hormone. +Acetyl CoA Bile acid. Acetoacetyl CoA Cytoplasmic HMG CoA +Acetyl CoA Synthase. HMG CoA HMG CoA RLE Reductase. Mevalonate Isoprenoid Unit (5C) Bile acid synthesis : Site : Liver & intestine. Cholesterol 7 α Hydroxylase(RLE) Liver 7 OH Cholesterol Cholic Acid Chenodeoxycholic Acid 1˚Bile acids: Intestine Enterohepatic 2˚Bile acids: Deoxycholic Acid Lithocolic Acid Circulation Lipoproteins : 01:08:33 Electrophoretic pattern of plasma Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 4 04 29 Chylomicrons VLDL LDL HDL ----- Active space ----- Source Intestine Liver VLDL (LP cascade Liver & intestine pathway) Composition Max TAG High TAG Maximum Cholesterol Lowest TAG. Lowest cholesterol Low cholesterol Low TAG High cholesterol. Functions Carries exogenous Carries Delivers cholesterol Delivers cholesterol from (dietary) TAG to endogenous TAG to peripheral tissues peripheral tissues to peripheral tissues. to peripheral and back to liver. liver for elimination. tissues. Apolipoproteins Apo B48 : Apo B100, Apo B100 Apo AI, Apo C, (Unique to chylomicron) Apo CII, Apo D, Apo CII, Apo E Apo E Apo E Characteristics Minimum density. Maximum density Maximum size. Minimum size. Remains at Origin Fastest electrophoretic mobility Hyperlipoproteinemias : Disease Molecular De- Serum levels fect Type 1 Familial Chylomicronemia Syndrome : Lipoprotein CM > VLDL Lipase TAG Apo C11. Cholesterol = Normal Type I1A Familial Hypercholesterolemia/ LDL receptor LDL ; Cholesterol FDB(Familial Defective apo B) ApoB-100 TAG normal Sitosterolemia ABCG5 & ABCG8 Secretes out plant sterol (Also a type of type 11 hyperlipoproteinemia) (not normal in animals) LDL Type 111 Apo E TAG Familial combined hyperlipoproteinemia Cholesterol (Broad ß disease/ Remnant removal disease) Hypolipoproteinemias : Disease Molecular Defect Serum levels Abetalipoproteinemia Microsomal Triglycerides Nascent CM transfer proteins Nascent VLDL,IDL,LDL Tangier’s disease Mutation ABCA-1 Fish eye disease Partial LCAT deficiency Norum’s disease Complete LCAT deficiency Biochemistry Revision v1.0 Marrow 6.5 2023  30 04 Biochemistry ----- Active space ----- Type 1 hyperlipoproteniemias : Familial chylomicronemia syndrome. Eruptive xanthoma Lipemia retinalis Milky white plasma/ strawberry coloured Type 11 hyperlipoproteniemias : Familial hypercholesterolemia. Corneal arcus Xanthomas Achilles tendon Type 111 hyperlipoproteinemias : Xanthoma Familial dysbetalipoproteinemia Tubero eruptive xanthoma Palmar xanthoma (bunch of grapes) (yellowish discoloration of palmar creases). Tangier’s disease : Yellowish/orange tonsil Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 5 05 31 BIOCHEMISTRY REVISION 5 ----- Active space ----- Amino Acid (AA) chemistry : amino H acid NH2- C -COOH α Most AA are α-AA. R variable side chain Classification of amino acids 00:00:43 I. Based on side chain : Sl No. Side chain AA 1. Aliphatic Simple Glycine, Alanine Branched Leucine, Isoleucine, Valine 2. OH group Serine, Threonine, Tyrosine 3. Sulphur group Cysteine, Methionine 4. Acid group (-COOH) Aspartic acid, Glutamic acid 5. Amide group (-CO-NH2) Asparagine Glutamine 6. Basic (-NH2) Histidine (aromatic), Arginine, Lysine 7. Aromatic Benzene ring Phenylalanine Phenol ring Tyrosine Indole ring Tryptophan 8. Imino acid (pyrrolidine ring) Proline II. Based on side chain characteristics : Polar Non polar Charged Uncharged 1. Branched chain AA. 1. Acidic AA : 1. Amides : 2. Aromatic AA : Aspartic acid. Asparagine, Glutamine. Tyrosine & Tryptophan. Glutamic acid. 2. OH group : Phenylalanine. 2. Basic AA : Serine, Threonine. 3. Simple AA : Alanine. Histidine. 3. Simple AA : Glycine. 4. Sulphur group : Arginine. 4. Sulphur group : Cysteine. Methionine. Lysine. Biochemistry Revision v1.0 Marrow 6.5 2023  32 05 Biochemistry ----- Active space ----- III. Based on metabolic fate : Purely ketogenic Both glucogenic and ketogenic Leucine, Lysine. Phenylalanine, Isoleucine, Tyrosine, Tryptophan. IV. Based on nutritional requirement (Essential amino acids) Methionine. Valine. Phenylalanine. Threonine. Isoleucine. Lysine. Tryptophan. Leucine Histidine. Note : All other AAs are non-essential. Arginine (semi-essential). Other amino acids : Selenocysteine : 21st protein forming amino acid. Enzymes containing Stop codon : UGA. selenocysteine : Precursor AA : Serine. i. Glutathione peroxidase. Formed by cotranslational modification. ii. Thioredoxin reductase. Pyrolysine : iii. Deiodinase. 22nd protein forming AA. iv. Selenoprotein p. Stop codon : UAG (recoding). Formed by cotranslational modification of precursor AA : Lysine. Properties of amino acids 00:09:31 1. Exhibits D & L isomerism (L isomers are present in proteins) (Except glycine which is optically inactive). 2. Aromatic AA absorb UV light (250 -290 nm) d/t presence of conjugate rings. 3. Exhibits buffering capacity : 1. Maximun when pH = pKa. 2. Maximum : Imidazole group of histidine (pKa = 6.4). 4. Zwitterions/ampholyte : pH = pI (isoelectric pH). Amino acids to proteins 00:13:06 H H H H NH3+- C -COOH + NH3+- C -COOH NH3+- C -CO NH - C -COOH R R R R Peptide bond : Uncharged & Polar. Partial double bond. “ Trans “ in nature. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 5 05 33 Structure of proteins : ----- Active space ----- Primary Linear sequence of AA. structure Peptide bonds (covalent) are involved. Not lost on denaturation. Secondary α helix : structure Right handed spiral. M/c & most stable structure. Intrachain hydrogen bond. β sheet/β pleated : 2nd m/c. Zig zag/extended structure. Interchain hydrogen bond. Parallel/anti-parallel. Tertiary 3D organization of single polypeptide. structure Domain : Structure that performs task. 1. Rossmann fold : NAD (P)+ binding domain present in oxidoreductase enzyme. 2. Immunoglobulin fold. Quaternary > 1 polypeptide. structure Subunit interaction. Note : Secondary structure has turns (short segments) & loops (long segments). Amino acid that disrupts alpha helix : Proline. Amino acid that induces bends in alpha helix : Glycine. Motifs are supersecondary structures. Protein folding 00:19:42 Auxillary proteins that assist protein folding : Molecular chaperones (heat shock proteins). Eg : BiP (Immunoglobulin heavy chain binding protein). Glucose Regulated Protein (GRP-94). Calreticulin. Calnexin. Calcium binding proteins Biochemistry Revision v1.0 Marrow 6.5 2023  34 05 Biochemistry ----- Active space ----- Protein misfolding diseases Prion diseases Prion related protein diseases Amyloidosis Prion related protein diseases : 1. Alzheimer’s disease. 2. Parkinson’s disease. 3. Beta thalassemia. 4. Cystic fibrosis. 5. Huntington’s disease. 6. Fronto Temporal Dementia (FTD). 7. Amyotrophic Lateral Sclerosis (ALS). 8. Dementia with Lewy Bodies (DLB). Protein degradation : Lysosomal degradation Proteasomal degradation ATP independent process ATP dependent process Long-lived proteins Short-lived proteins Membrane proteins Misfolded proteins Note : Proline (P) glutamate (E) Serine (S) Threonine (T) : PEST. PEST sequence is needed for proteasomal degradation. PEST + Ubiquitin → protein degradation. Process is called “ kiss of death “. Collagen 00:24:56 Most abundant protein in human. Most abundant extracellular protein. Major type of collagen in bone : Type I. Most abundant type of collagen : Type I. Major type of collagen in cartilage : Type II. Major type of collagen in microfibrils : Type VI. Major type of collagen in basement membrane of kidney : Type IV. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 5 05 35 Structure of collagen : ----- Active space ----- ( Gly-X-Y..... Gly-X-Y)1000 X - Hydroxyproline. → → Y - Hydroxylysine. Most abundant amino acid : Glycine. 3 Alpha domains Recurring amino acid : Glycine. Alpha chain turn in right handed Triple helix Left handed right handed direction Quarter Staggered structure (side to side Triple helix : 1/4th distance) : 1/4th 1/4th lysyl & hydroxylysyl Lysyl oxidase Covalent cross linking sed tensile strength residues Copper Synthesis of collagen : Intracellular Extracellular Place RER of Fibroblast Extracelluar matrix Product Procollagen Tropocollagen formed Major 1. Hydroxylation : 1. Cleavage. events Prolyl & lysyl hydroxylase. 2. Quarter staggered Requires Vit-C. structure Facilitates triple helix. 3. Covalent cross links : By 2. Glycosylation : Aldol condensation (requires Hydroxylysyl residues. Cu). 3. Triple helix formation : 3α chains join Biochemistry Revision v1.0 Marrow 6.5 2023  36 05 Biochemistry ----- Active space ----- Diseases associated with collagen : Type of collagen Disease associated affected Type 1 Osteogenesis imperfecta Osteoporosis EDS type VII Type II Severe chondrodysplasias Osteoarthritis Type III EDS type IV Type IV Alports syndrome Type VI Bethlem myopathy Type VII Epidermolysis bullosa, dystrophic Type 10 Schmid metaphyseal dysplasia Applied aspects - General amino acid metabolism 00:35:24 Ketoacids Biologically Amino acid imp Amines NH3 CO2 Handling of amino group : I. Transamination : α Amino acid α keto acid PLP / B6 Transaminase α keto acid α Amino acid Alanine α ketoglutarate Aspartate α ketoglutarate PLP / B6 ALT PLP / B6 AST Pyruvate Glutamate Oxaloacetate Glutamate Site : Cytoplasm. Characteristics : Reversible. Concentrate all amino group in AA as glutamate (non toxic). Ping pong mechanism. AST & ALT : Specific for substrate AA, non specifc for alpha keto glutarate. Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 5 05 37 II. Transport of amino group : ----- Active space ----- any α Amino acid α ketoglutarate any α keto acid Glutamate Glutamine First line Purine synthetase +NH3 pyramidine trapping of Porphyrines ammonia Glutamine Note : Glutamine is transport form of ammonia in most organs except in muscle (alanine). Glutamic acid on decarboxylation : GABA. Glutamic acid on deamination : Alpha Ketoglutarate. III. Oxidative deamination : Liver Alanine Glutamate Glutamine Glutamate Dehydrogenase NH3 Urea cycle NAD(P) + NAD(P)H NOTE : α ketoglutarate Liver GDH is allosterically : Activated by : ADP. Inhibited by : ATP, GTP & NADH. Urea/ornithine cycle/Kreb’s Henseleit cycle 00:43:51 Organ : Liver. Organelles : Cytoplasm, mitochondria. Respiratory CO2 NH2- CO -NH2 Aspartate from TCA NH3 Biochemistry Revision v1.0 Marrow 6.5 2023  38 05 Biochemistry ----- Active space ----- Mitochondria Cytoplasm Co + NH3 2 2 ATP Aspartate CPS-I (RLS) 2 ADP Carbamoyl Phosphate “CITRIN” OTC Citrulline Ornithine Citrulline + Aspartate I ATP Ornithine Transporter 2 Pi Argininosuccinate Synthase I AMP Argininosuccinate [AS] Ornithine H 2O AS lyase Urea Arginase Arginine Fumarate TCA cycle Rate limiting step (RLS) : CPS I. Allosteric activators of above step : N - Acetyl glutamate. Urea cycle disorders : Disease Enzyme defect Hyperammonemia type 1 CPS- 1 (Carbamoyl Phospate Synthetase) Hyperammonemia type II OTC (Ornithine Transcarbamylase) HHH syndrome Ornithine transporter (Hyperammonemia Hyperornithinemia Homocitrullinemia) Citrullinemia type I Arginiosuccinate synthetase Citrullinemia type II Citrin transporter Argininosuccinic aciduria Arginiosuccinate Lyase Argininemia Arginase Biochemistry Revision v1.0 Marrow 6.5 2023  Biochemistry Revision 5 05 39 Hyperammonemia type 2 : M/c urea cycle defect. ----- Active space ----- Carbamoyl phosphate Defect in OTC accumulation Seeps into cytoplasm Orotic aciduria Orotic acid synthesis Excretion of Pyrimidine synthesis pyrimidine in urine Diagnosis of urea cycle disorders : Blood NH3 Check blood pH Increased Decreased Urea cycle D/o Organic aciduria Specific AA Non specific AA Plasma orotic Acid Citrulline : Citrullinemia. Increased Normal Ornithine : HHH syndrome. Arginine : Argininemia. Hyperammonemia Hyperammonemia Arginosuccinate : Arginino- type 2 type 1 succinic aciduria. Note : AA that do not undergo transamination : a. Proline. b. Hydroxyproline. c. Threonine. d. Lysine. Amino acid that increases in plasma during starvation : Alanine. AA that increases in plasma during hyperammonemia : Glutamine. Biochemistry Revision v1.0 Marrow 6.5 2023  40 06 Biochemistry ----- Active space ----- BIOCHEMISTRY REVISION