Enzymes & isoenzymes by Dr. Anurag Yadav

Medical Biochemistry

Biochemistry of Enzymes
Classification of Enzymes
Enzyme Inhibition
Isoenzymes
Enzyme Inhibition
Clinical Enzymology

1. ENZYMEs MNR MEDICAL COLLEGE & HOSPITAL Dr Anurag Yadav MBBS, MD Assistant Professor Department of Biochemistry Instagram page –biochem365 YouTube – Dr Biochem365 Email: [email protected]

2. OBJECTIVE What are enzymes? Definitions Characteristics of enzymes Classification of enzymes

3. 31-07-2020 3 Berzelius History

4. 31-07-2020 4 Kunhe History

5. What Are Enzymes? Enzymes are biocatalysts 7/31/2020 5

6. BIOCHEMICAL NATURE OF ENZYME All enzymes are proteins except ribozymes. They are distinguished from other proteins by catalytic action. The catalytic power of enzyme is d/u primary, secondary, tertiary & quaternary structure of the protein molecule. Change can affect enzyme activity.

7. Chemical Nature of Enzymes They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. Enzymes can be denatured and precipitated with salts, solvents and other reagents.

8. Characteristics Catalysts for biological reactions Most are proteins Lower the activation energy Increase the rate of reaction Heat labile Can be precipitated by protein precipitating agents May contain cofactors such as metal ions or organic (vitamins) 8

9. Substrate The substance upon which an enzyme acts. Product The enzyme will convert the substrate into product. 7/31/2020 9

10. Enzymes Made of protein Present in all living cells Converts substrates into products Biological catalysts Increase the rate of chemical reactions Remain unchanged by chemical reaction Characteristics of enzymes

11. Name of Enzymes End in – ase Identifies a reacting substance sucrase – reacts sucrose lipase - reacts lipid Describes function of enzyme oxidase – catalyzes oxidation hydrolase – catalyzes hydrolysis Common names of digestion enzymes still use – in Pepsin, Trypsin 11

12. Enzyme classification IUBMB System of Classification Described by International Union of Biochemistry and Molecular Biology (IUBMB) in 1964

13. CLASSIFICATION (IUBMB) OXIDOREDUCTASES TRANSFERASES HYDROLASES LYASES ISOMERASES LIGASES

14. Enzyme Code number “EC” Enzyme Code number 1 st digit - main class. 2 nd digit – subclass. Type of group involved. 3 rd digit – sub-sub class 4 th digit – number given to the enzyme in sub-sub class E.g. EC(1.1.1.27)

15. Creatine kinase

16. Classification of Enzymes Class Reactions catalyzed 1. Oxidoreductoases O xidation-reduction 2. Transferases Transfer group of atoms 3. Hydrolases Hydrolysis-cleave & add water 4. Lyases Cleave without adding water 5. Isomerases R earrange atoms 6. Ligases C ombine molecules using ATP 17

17. EC-1 OXIDOREDUCTASE Enzymes involved in oxidation- reduction reactions. Catalyze the electron transfer . Oxygen- oxidases. Hydrogen- dehydrogenases. Alcohol Dehydrogenase Alcohol + NAD + Aldehyde + NADH + H +

18. Class 1: ENZYME SUBSTRATE PRODUCT Lactate dehydrogenase Lactate Pyruvate Xanthine oxidase Xanthine Uric acid L Amino acid oxidase D amino acids Keto acids Cytochrome oxidase Reduced Cytochrome C Oxidized Cytochrome-C Alcohol dehydrogenase Alcohol Aldehyde

19. EC-2 TRANSFERASES Catalyze the transfer of functional groups. (amino, carboxyl, methyl, phosphoryl, etc ) (A-X) +B A+(B-X)

20. A . METHYL group---  e.g. Transmethylase B. ALDEHYDE or KETONIC group e.g. Transaldolase or transketolase . C. ACYL GROUP e.g.Aceyltransferase D . AMINO-KETO GROUP- Aminotransferase E. KINASES are specialized transferase that regulate metabolism by transferring phosphate from ATP to other molecules e.g. Hexokinase      ATP +Glucose -----  G-6-P+ ADP

21. EC-3 HYDROLASES That bring about hydrolysis of compounds. Catalyze the cleavage of C-O, C-N, C-C, etc by adding water A-B + H 2 0 A-OH + B-H Glucose-6-phoshate+H 2  Glucose + Pi glucose-6-phosphatase

22. 1. LIPASES--- e.g.Phospholipases , Lipoprotein lipase 2. PHOSPHATASES------e.g.Glucose-6-Phosphatase 3. CHOLINE ESTERASE -hydrolysases acetylcholine 4. PEPTIDASES-----hydrolyses peptides 5. NUCLEASE --- e.g.nucleotidase , nucleosidase 6. Which break CARBOHYDRATES e.g. Amylase act on amylose Lactase, Maltase 7. Enzymes acting on C—N linkage --- Urease, Asparginase. Glutaminase, Arginase  

23. EC-4 LYASES Catalyze the cleavage of C-O, C-C & C-N bonds by means other than hydrolysis, giving rise to compound with double bonds. A-X LYASE A │ ║ + X-Y B-Y B Ex- Aldolase , Decarboxylase, Carbonic Anhydrase, Cysteine Desulfurase , HMG Co-A Lyase

24. EC-5 ISOMERASES Catalyze intramolecular (structural or geometric) changes in a molecule. ABC CAB glucose,6,phosphate Fructose,6,phosphate Phoshohexose isomerase

25. Triose Phosphate Isomerase

26. MUTASE

27. EC-6 LIGASES (Synthetases) Catalyze the joining of two molecules coupled with the hydrolysis of pyrophosphate bond of ATP. A + B + ATP AB + ADP +Pi Glutamate+ NH 3 + ATP Glutamine synthatase Glutamine + ADP+Pi

28. GLUTAMINE SYNTHETASE

29. BIOTIN CARBOXYLASE

30. ZYMOGEN OR PROENZYME Enzymes which are present in inactive form, which must be cleaved to be activated Blood & digestive tract- Enzymes present in precursor form. E.g. Chymotrypsinogen. Prothrombin. Proelastase Their synthesis in proenzyme form prevent them from catalyzing reactions in the cell where they are synthesized .

31. Co-enzymes E nzyme may be simple protein or complex protein containing protein part (Apo-enzyme) + Holoenzyme non-protein part (Co-enzyme) Metallo -enzymes: enzymes which requires metal ions for their activity. Ex: magnesium for hexokinase Co-factors: Co- enzyme+Metal ion

32. Features of Co-enzymes E ssential for the biological activity of the enzyme Co-enzyme is a low molecular weight organic Substance It is heat stable . C ombine loosely with the enzyme molecules when the reaction is completed, the co-enzyme is released from the apo -enzyme, and can bind to another enzyme molecule Most of the co-enzymes are derivatives of vitamin B complex group of substances 31-07-2020 33

33. Co-enzymes may be divided into two groups Those taking part in reactions catalyzed by oxidoreductases by donating or accepting hydrogen atoms or electrons Those co-enzymes taking part in reactions transferring groups other than hydrogen 31-07-2020 34

34. CLASSIFICATION For transfer of hydrogen : NAD + , NADP + , FMN, FAD, Lipoic acid, Coenzyme Q. For transfer of group other than hydrogen Co-A-SH, Thiamin pyrophosphate, Pyridoxal phosphate, Tetrahydro folate , Biotin, Methyl cobalamine Deoxy adenosyl cobalamine

35. Dietary precursor Coenzymes Group transfer Thiamin (B 1 ) Thiamine pyrophosphate Aldehyde Nicotinic acid (Niacin ) Nicotinamide adenine dinucleotide Hydride(H + ) Riboflavin (B 2 ) Flavin adenine dinucleotide Electron Panthothinic acid Coenzyme-A Acyl group Pyridoxine Pyridoxial phosphate Amino group

36. Metalloenzymes Metals enzymes Fe 2+ OR Fe 3+ Cytochrome oxidase Catalase, perxidase Cu 2+ Cytochrome oxidase, SOD Zn 2+ Carbonic anhydrase Alcoholic dehydrogenase Mg 2+ Hexokinase Glucose-6-phosphatase Pyruvate kinase Mn 2+ Arginase Ribonucleotide reductase

37. LOCALIZATION OF ENZYMES Enzymes are located either -Intracellularly or -Extracellularly. Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze the reactions of metabolic pathways. Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals E nzymes of the circulatory system are responsible for regulating the clotting of blood Almost every significant life process is dependent on enzyme activity.

38. Active site

39. 31-07-2020 41 Specificity of Enzyme Action

40. Specificity of Enzyme A ction T he ability of an enzyme to discriminate b/w two competing substrates. Significance: specificity makes it possible for a number of enzymes to co-exit in the cell without interfering in each other’s actions. TYPES: - Absolute specificity - Group specificity - Reaction specificity - Bond specificity - Stereo specificity

41. Absolute specificity

42. Group specificity One enzyme can catalyse the same reaction on a group of structurally similar compounds Ex- Hexokinase can catalyse phosphorylation of glucose , galactose and mannose Lipase cleaves Various groups of Lipids

43. Reaction specificity Almost only one enzyme catalyzes a given specific reaction

44. Most of the proteolytic enzymes are showing group (bond ) specificity Ex- proteolytic enzymes Exopeptidases Endopeptidases -hydrolyzing terminal -centrally located peptide bond peptide bond - carboxypeptidase -pepsin, trypsin, - aminopeptidase - chymotrypsin Bond specificity

45. Stereo specificity Many enzymes show specificity towards stereoisomers. They act on only one type of isomer E.g : L-lactate dehydrogenase will act only on L- lactic acid and not D- lactic acid

46. Mechanism of action of enzymes

47. How do enzymes Work? Enzymes work by Lowering of Activation Energy 7/31/2020 49

48. Enzymes accelerate reaction rate by providing transition states with low activational energy for formation of products Hence reaction rate is enhanced by many folds in the presence of enzymes The total energy of the system remains the same and equilibrium state is not disturbed

49. Theories to explain enzyme substrate interaction Michaelis-Menten Theory Fischers Template Theory Koshland’s Induce F it Theory

50. MICHAELIS–MENTEN THEORY In 1913 put forward the Enzyme–Substrate complex theory The enzyme (E) combines with the substrate (S), to form an enzyme-substrate (ES ) complex, which immediately breaks down to the enzyme and the product (P) E + S → E–S Complex → E + P

51. Ex - P hosphatase Glucose-6-P → Glucose + Pi The active center of this enzyme contains a serine residue a. E-Serine-OH+Glucose-6-P → E-Serine-O-P+Glucose b. E-Serine-O-P → E-Serine-OH+Pi

52. FISCHER'S TEMPLATE THEORY Lock and K ey Model It states that the three dimensional structure of the active site of the enzyme is complementary to the substrate Enzyme and substrate fit each other

53. KOSHLAND'S INDUCED FIT THEORY The substrate induces conformational changes in the enzyme , such that precise orientation of catalytic groups is effected Allosteric regulation can also be explained by the hypothesis of Koshland

54. ENZYME KINETICS Enzyme kinetics  is the study of the chemical reactions that are catlysed by  enzymes . In   enzyme kinetics , the reaction rate is measured and the effects of various conditions of the reaction are investigated

55. Velocity or rate of enzyme reaction is assessed by the rate of change of substrate to product per unit time The velocity is proportional to the conc. of reacting molecules. A + B -------------------------→ C + D V α [ A] [B]

56. The equilibrium constant of the reaction is the ratio of reaction rate constants of forward and backward reactions Forward reaction R1 = K1 [A] [B] Backward reaction R2 = K2 [C] [D] At equilibrium, R1 = R2 Or, K1 [A] [B] = K2 [C] [D ]

57. FACTORS AFFECTING ENZYME ACTIVITY 1. Enzyme concentration 2. Substrate concentration 3. Product concentration 4. Temperature 5 . Hydrogen ion concentration (pH) 6. Presence of activators 7. Presence of inhibitors 8. Presence of repressor or derepressor 9. Covalent modification

58. 1. Enzyme Concentration i . Rate of a reaction or velocity (V) is directly proportional to the enzyme concentration

59. 3. Effect of Concentration of Products when product concentration is increased, the reaction is slowed, stopped or even reversed E1 E2 E3 A -------→ B ----------→ C -------||------→ D

60. 4. Effect of Temperature

61. The temperature coefficient (Q10) is the factor by which the rate of catalysis is increased by a rise in 10°C . T he rate of reaction of most enzymes will double by a rise in 10°C.

62. 5. Effect of pH

63. Enzymes have the optimum pH between 6 and 8. Exceptions are pepsin (with optimum pH 1-2); alkaline phosphatase (optimum pH 9-10) and acid phosphatase (4-5).

64. 2. Effect of Substrate Concentration As substrate concentration is increased , the velocity is also correspondingly increased in the initial phases; but the curve flattens afterwards The maximum velocity obtained is called Vmax

65. Substrate Saturation of an Enzyme A. Low [S] B. 50% [S] or K m C. High, saturating [S]

66. Michaelis Constant (Km) Michaelis theory, the formation of enzyme – substrate complex is a reversible reaction, while the breakdown of the complex to enzyme + product is irreversible

67. The Michaelis-Menten equation It is relationship between initial reaction velocity vi and substrate concentration [S ] Km is Michaelis-Menten constant

68. When Vo = ½ Vmax Km = [S] Km value is substrate concentration at half-maximal velocity

69. Salient Features of Km 1. Km value is substrate concentration ( expressed in moles/L) at half-maximal velocity 2. Km is independent of enzyme concentration 3. It is the Signature of the enzyme 4. It denotes the affinity of the enzyme towards the substrate Low Km - high Affinity for substrate High Km –low affinity for substrate

70. Useful to compare Km for different substrates for one enzyme Hexokinase : D-fructose – 1.5 mM D-glucose – 0.15 mM Useful to compare Km for a common substrate used by several enzymes Hexokinase: D-glucose – 0.05 mM Glucokinase : D-glucose – 10 mM

71. Uses of K m Experimentally, K m is a useful parameter for characterizing the number and/or types of substrates that a particular enzyme will utilize It is the K m of the rate-limiting enzyme in many of the biochemical metabolic pathways that determines the amount of product and overall regulation of a given pathway

72. Limitations of Michaelis-Menten equation Low [S] have to be used to plot the initial segment where Vo cannot be measure precisely Very high [S] required to to draw a the final platue When the points observed for velocity are too scattered the hyperbolic graph cannot be drawn precisely It is difficult to extrapolate the hyperbolic graph if required

73. The plot provides a useful graphical method for analysis of the  Michaelis-Menten  equation: Taking the reciprocal gives V  is the reaction velocity (the reaction rate) K m  is the  Michaelis–Menten constant V max  is the maximum reaction velocity [ S ] is the substrate concentration 80

74. Lineweaver –Burk Plot or Double Reciprocal Plot

75. ENZYME INHIBITION Enzyme inhibitors -are molecular agents that interfere with catalysis; slowing or halting enzymatic reactions. There are two broad classes of enzyme inhibitors: reversible and irreversible

76. Reversible Inhibition- Reversible Inhibition –activity of enzyme is fully restored when inhibitor physically removed from system.

77. COMPETITIVE INHIBITOR : competes with the substrate for the active site of an enzyme.

78. Examples: Sulphonamide (PABA) – pteroid synthetase Dicoumarol -- Vitamin k epoxide reductase Lovastatin -- HMG Co reductase Allopurinol – Xanthine oxidase Methotrexate – Dihydrofolate reductase

79. NONCOMPETITIVE INHIBITION Binding of the inhibitor does not affect binding of substrate. No competition between substrate & inhibitor. Formation of both EI and EIS complexes is therefore possible.

80. Examples Trypsin inhibitors in Soyabean & raw egg Parasite Ascaris contains pepsine & trypsine inhibitors

81. IRREVERSIBLE INHIBITION The irreversible inhibitors - Bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity,

82. Examples Cyanide inhibits cytochrome oxidase. Fluoride will inhibit the enzyme , enolase , and consequently the glycolysis . Iodoacetate inhibits enzymes having-SH group in their active centers . BAL (British Anti Lewisite; dimercaprol ) is used as an antidote for heavy metal poisoning . The heavy metals act as enzyme poisons by reacting with the SH group. BAL has several SH groups with which the heavy metal ions can react and thereby their poisonous effects are reduced

83. Effect of inhibitors… Type of inhibitor Km Vmax Irreversible No effect Decreased Reversible competitive Increased No effect Reversible noncompetitive No effect Decreased Reversible uncompetitive Decreased Decreased

84. Increasing the substrate concentration will abolish the competitive inhibition , but will not abolish noncompetitive inhibition

85. Suicide inhibition It is a special type of irreversible inhibition of enzyme activity. It is also known as mechanism based inactivation. The inhibitor makes use of the enzyme's own reaction mechanism to inactivate it (mechanism based inactivation ). the structural analog is converted to a more effective inhibitor with the help of the enzyme to be inhibited. This new product irreversibly binds to the enzyme and inhibits further reactions

86. Examples: DFMO ( difluromethylornithine ) in treatment of trypanosomia . Allopurinol in gout Aspirin as anti-inflammatory.

87. of

88. Competitive inhibition Therapeutic agent Enzyme inhibited Clinical use Acetazolamide Carbonic anhydrase Diuretic Methotrexate Folate reductase Anti cancer Captopril Angiotensin converting enzyme Hypertension Statins HMG CoA reductase Hypercholesterolemia Allopurinol Xanthine oxidase Gout Dicoumarol Vit K epoxide reductase Anti coagulant Sulphonamide Pteroid synthetase Antibiotic Acyclovir DNAP of virus antiviral

89. Competetive inhibitors Azaserine Phosphoribosyl amidotransferace Anti cancer Cytosine arabinoside DNA polymerase Anti cancer Neostigmine Ach esterase Myasthenia gravis Osteltamivir Neuraminidase Influenza Penicillin Transpeptidase Anti bacterial Isonicotinic acid hydrazide Anti tubercular

90. Irreversible Inhibitors Therapeutic agent Enzyme inhibited Clinical use Cyanide Cytochrome oxidase Fluoride Enolase Glycolysis inhibition BAL ( dimercaprol ) Thiol group enzymes Heavy metal poisoning Iodoacetate SH group containing enzymes Heavy metal poisoning Suicide inhbition Allopurinol Xanthine oxidase Gout MAO inhibitors ( deprenyl ) Mono amine oxidase Mood stabilizers, antidepressant .

91. Enzyme regulation

92. Enzyme regulation The facility to increase or reduce the rate of an enzyme catalysed reaction is a crucial part of metabolic control and therefore the adaptability of metabolism as this allows optimal utilization of possibly scarce resources. In short, a cell must be able to control its metabolic activities in order to meet a challenge from the environment.

93. Enzyme regulation … Rate limiting step of a metabolic pathway is that reaction which determines the rate and direction of the entire pathway

94. Criteria for rate limiting enzyme Regulated enzyme its activity and /or synthesis should be regulated in vivo Rate limiting step is catalysed practically unidirectionally or irreversible by the enzyme in vivo Determines the direction of the entire pathway Usually the initial step of a pathway so that the intermediates of earlier steps would not accumulate in case of feed back inhibition or repression of the rate limiting enzyme.

95. Pathways Rate limiting enzymes Adipose tissue lipolysis Hs triacylglycerol lipase Cholesterol synthesis HMG CoA reductase TCA cycle alpha KG dehydrogenase Fatty acid synthesis Acetyl CoA carboxylase Gluconeogenesis Fructose 1,6 bisphosphatase , PEP caboxylase Glycogenolysis Glycogen phosphorylase Glycogenesis Glycogen synthase Urea synthesis Carbamoyl phosphate synthase Pentose phosphate pathway Glucose 6 phosphate dehydrogenase Purine biosynthesis PP ribosyl - P glutamyl amidotransferase

96. Enzyme regulation types

97. Covalent modification Irreversible covalent modification Activation of inactive proenzymes or zymogens by the action of partial Proteolysis (hydrolysis). Ex: trypsinogen , chymotrypsinogen , pepsinogen , proinsulin , clotting factors, procollagen

98. Covalent modification Reversible covalent modification This is by the process of phosphorylation of proteins on seryl , threonyl , or tyrosyl residues, catalyzed by protein kinases , is thermodynamically spontaneous. Equally spontaneous is the hydrolytic removal of these phosphoryl groups by enzymes called protein phosphatases .

99. Feedback regulation Feedback regulation, a phenomenologic term devoid of mechanistic implications Ex: D ietary cholesterol decreases hepatic synthesis of cholesterol Regulation in response to dietary cholesterol involves curtailment by cholesterol or a cholesterol metabolite of the expression of the gene that encodes HMG- CoA reductase (enzyme repression)

100. Allosteric Enzymes An important group of enzymes that do not obey Michaelis - Menten kinetics comprises the allosteric enzymes. These enzymes consist of multiple subunits and multiple active sites.

101. The activity of an allosteric enzyme may be altered by regulatory molecules that are reversibly bound to specific sites other than the catalytic sites. The catalytic properties of allosteric enzymes can thus be adjusted to meet the immediate needs of a cell. Allosteric enzymes are key regulators of metabolic pathways in the cell.

102. Allosteric modulation The binding of substrate to one active site can affect the properties of other active sites in the same enzyme molecule. A possible outcome of this interaction between subunits is that the binding of substrate becomes cooperative : positive allosteric effect

103. Allosteric modulation Negative cooperativity , in which the binding of substrate to one active site decreases the affinity of other sites for substrate Negative allosteric modulation (also known as  allosteric inhibition) For example, when  2,3-BPG  binds to an allosteric site on hemoglobin , the affinity for oxygen of all subunits decreases

104. Allosteric modulators Enzymes Activators Inhibitors Acetyl CoA caboxylase Citrate Palmitoyl CoA Aspartate transcarbamoylase ATP CTP Carbamoyl phosphate synthase (mitochondria) N acetyl glutamate (cytoplasm) PP ribose P, ATP UMP, UDP, UTP, CTP Fructose 1,6 bisphosphate Fructose 2,6 bisphosphate Glycogen synthase Glucose 6 phosphate Phosphofructokinase 1 Fructose 2, 6 bisphosphate ATP Pyruvate carboxylase Acetyl CoA

105. Compartmentalization Pathways in eukaryotic cells are often compartmentalized within cytoplasmic organelles by intracellular membranes. Thus we find particular pathways associated with the mitochondria, the lysosomes , the peroxisomes , the endoplasmic reticulum

106. Compartmentalization Enzymes that degrade proteins and polysaccharides reside inside lysosomes Fatty acid biosynthesis occurs in the cytosol , whereas fatty acid oxidation takes place within mitochondria

107. Induction Induction is effected through the process of derepression . The inducer will relieve the repression on the operator site and will remove the block on the biosynthesis of the enzyme molecules . Tryptophan pyrrolase and transaminases are induced by glucocorticoids . Glucokinase is induced by insulin . ALA synthase is induced by barbiturates.

108. Repression repressor acts at the gene level. Whereas i nhibition at enzyme level. key enzyme of heme synthesis, ALA synthase is autoregulated by heme by means of repression

109. DIAGNOSTIC ENZYMOLOGY

110. ENZYMES OF DIAGNOSTIC IMPORTANCE ENZYMES TISSUE ORIGIN CLINICAL SIGNIFICANCE 1. Acid phosphatase Prostate, RBC Ca prostate 2. ALT Liver ,Muscle , heart liver disease 3. ALP Brain, Liver Bone & Hepatobiliary D 4. Amylase Pancreas Pancreatic disease 5. AST Heart, Liver MI, Hepatitis 6. Aldolase Skeletal muscle Muscular dystrophy 7. Cholinesterase Liver OP poisoning 8. Creatine kinase SM, Heart MI, Muscular dystrophy 9. GGT Hepatobiliary sys Hepatobiliary D, Alcohol 10. LDH Heart, Liver ,SM, RBC MI, Hemolysis 11. 5’-NTS Hepatobiliary tract Hepatobiliary disease 12. Prostate specific Ag Prostate Ca prostate 13. Lipase Pancreas Pancreatitis 14. Trypsin Pancreas Cystic fibrosis

111. ENZYME PATTERNS IN DISEASES Hepatic disease – ALT , AST, NTP, ALP, GGT Pancreatic diseases - Amylase, lipase, Trypsin, & Chymotrypsin Myocardial infarction - CK-MB, AST, LDH

112. CARDIAC MARKERS SL NO ENZYMES START APPEARS PEAK LEVEL RETURNS NORMAL 1 Myoglobin 1 hr 6-12 hr 24 hr 2 Troponin - I 4-6 hr 14-24 hr 3-5 days 3 Troponin -T 6hrs 72hrs 7-14 days 3 CK-MB 3-6 hr 12-24 hr 2-3 days 4 AST/SGOT 6-12 hr 24-48 hr 4-5 days 5 LDH 2 8-16 hr 48-72 hr 7-12 days

113. CARDIAC MARKERS - MB

114. FEATURES OF LD SIOENZYMES ISOENZYME EP MOBILITY TISSUE OF ORIGIN %AGE IN SERUM LD-1 Fastest Heart, RBC, kidney 30% LD-2 Faster Heart, RBC kidney 35% LD-3 Fast Brain 20% LD-4 Slow Liver 10% LD-5 Slowest Skeletal muscle 5%

115. ENZYME PATTERNS IN DISEASES Hepatic disease – ALT , AST, NTP, ALP, GGT Pancreatic diseases - Amylase, lipase, Trypsin, & Chymotrypsin Myocardial infarction - CK-MB, AST, LDH

116. Hepatic disease TRANSAMINASES Normal ranges: ALT/SGPT – 10-40 IU/L AST/SGOT - 10-30 U/L

117. CONCENTRATION IN DIFF TISSUES ALT AST 10000 1000 100 10 1 Liver Heart SM Pancreas Serum

118. ALANINE + a KETOGLUTARATE PLP ALT PYRUVATE + GLUTAMATE ASPARTATE + a KETOGLUTARATE PLP AST OXALOACETATE + GLUTAMATE

119. AST Increased in parenchymal liver diseases Hepatitis, malignancies AST increased in MI

120. ALT High increase (300-1000) – Toxic Hepatitis, Viral Hepatitis Moderate increase (50-100) – Chronic liver disease, Cirrhosis, Hepatitis In carcinoma of liver 5-10 fold increase (AST & ALT) AST higher than ALT 7/31/2020 143

121. ENZYMES IN BILIARY TRACT DISEASES 5’-NTD GGT ALP

122. ALKALINE PHOSPHATASE Ecto enzyme - Cell membrane Metaloenzyme – Zinc Optimum PH – 9- 10 Present in Intestinal epithelial cells,bone osteoblasts , liver, kidney, placenta Normal range – 40-125 U/L Elevated Children's – 2.5 times Pregnancy

123. CLINICAL SIGNIFICANCE Moderate increase (2-3 Times) Alcoholic hepatitis Infective hepatitis High increase (10-12 Times) Obstructive jaundice - Gall Stone - Ca head pancreas Very High Levels (10-25 Times) -bone cancer - Paget's disease - Rickets - Healing bone #

124. 5’-Nucleotidase/Nucleotide phosphatase Ribonucleotide phosphohydralase Ecto-enzyme – present on cell membrane Marker enzyme for PM Optimum PH – 6.6 – 7 Normal level – 2-10 U/L

125. CLINICAL SIGNIFICANCE OF NTP Moderate elevation – Hepatitis Highly Elevated – Biliary obstruction

126. g - Glutamyl transferase GGT FUNCTIONS Transfer of AA’s from one peptide to another peptide Synthesis of glutathione Transport of aa across the cell membrane Location: -Liver, Kidney, Placenta Normal range : 10-30 U/L

127. CLINICAL SIGNIFICANCE More sensitive than ALP, NTP & AST, ALT Moderate increase – infective hepatitis Increased in Alcoholics – proportional to Alcohol intake Liver carcinoma increased earlier than other enzymes

128. CARDIAC MARKERS CK AST/SGOT LDH CK-MB CTn = TnI & TnT OLD CM NEW CM

129. CARDIAC MARKERS SL NO ENZYMES START APPEARS PEAK LEVEL RETURNS NORMAL 1 Myoglobin 1 hr 6-12 hr 24 hr 2 Troponin - I 4-6 hr 14-24 hr 3-5 days 3 Troponin -T 6hrs 72hrs 7-14 days 3 CK-MB 3-6 hr 12-24 hr 2-3 days 4 AST/SGOT 6-12 hr 24-48 hr 4-5 days 5 LDH 2 8-16 hr 48-72 hr 7-12 days

130. CARDIAC MARKERS - MB

131. CREATINE KINASE Creatine CK Creatine phosphate ATP ADP N Males -15-100U/L Females – 10-80U/L CK-MM – 80% CK-MB – 5% CK-BB – 1%

132. Clinical Significance CK-MB increased in MI CK-MM increased Mascular Dystrophies, Crush Injuries CK-BB increased in Cerebrovascular accidents

133. LACTATE DEHYDROGENASE PYRUVATE LDH LACTATE N- 100-200IU/L LDH levels are 100 times more inside the RBC than in the Plasma Hemolysis – false + ve reasults CLINICAL SIGNIFICANCE Increased in hemolytic anemia, hepatocellular damage, mascular dystrophy, carcinomas, leukemias , MI

134. ISOENZYMES ISOENZYME SUBUNITS TISSUE OF ORIGIN % IN SERUM LDH-1 H4 HEART 30% LDH-2 H3M1 RBC 35% LDH-3 H2M2 BRAIN 20% LDH-4 H1M3 LIVER 10% LDH-5 M4 SKELETAL MUSCLE 5%

135. LDH FLIPPED PATERN IN MI NORMAL MI

136. CARDIAC SPECIFIC TROPONINS Contractile proteins of all myofibrils

137. Troponin-I N- 1-10 microgm/L Not increased in muscle injury Troponin-T N- <50ngm/L

138. PANCREATIC ENZYMES Amylase Lipase PROENZYMES – Trypsinogen - Chymotrypsinogen

139. AMYLASE Amylase splits starch to dextrins , maltose Types – Salivary & Pancreatic Normal range : Serum - 50-120 U/L urine - < 375 U/L M W = 55,000 Optimum PH = 6.9 – 7 Calcium activates the enzyme

140. HYDROLYSIS OF STARCH

141. CLINICAL SIGNIFICANCE Acute pancreatitis: 1000 times increase Rise within 2-12 hr Peak – 12-72 hr Normal – 3-4 days Moderate increase – chronic pancreatitis, mumps, obstruction of pancreatic duct Urinary amylase increased in acute pancreatitis increased on 1 st day & remains increased for 7-10

142. LIPASE Hydrolyse Triglycerides Requires Colipase , bile salts N- 10-60U/L Location : - Pancreas Increased in acute pancreatitis Increases within 4-8hrs, peaks 24hrs, persists for 7-14 days

143. THERAPEUTIC ENZYMES SL NO ENZYMES APPLICATION 1 Asperginase ALL 2 Streptokinase Lyse clot on MI 3 Pepsin & trypsin Used in GI disorders 4 Fibrinolysin Used on wounds 5 α1-antitrypsin Emphysema 6 Collagenase Debridement of dermal ulcers/burns

144. ENZYMES OF DIAGNOSTIC IMPORTANCE ENZYMES TISSUE ORIGIN CLINICAL SIGNIFICANCE 1. Acid phosphatase Prostate, RBC Ca prostate 2. ALT Liver ,Muscle , heart liver disease 3. ALP Brain, Liver Bone & Hepatobiliary D 4. Amylase Pancreas Pancreatic disease 5. AST Heart, Liver MI, Hepatitis 6. Aldolase Skeletal muscle Muscular dystrophy 7. Cholinesterase Liver OP poisoning 8. Creatine kinase SM, Heart MI, Muscular dystrophy 9. GGT Hepatobiliary sys Hepatobiliary D, Alcohol 10. LDH Heart, Liver ,SM, RBC MI, Hemolysis 11. 5’-NTS Hepatobiliary tract Hepatobiliary disease 12. Prostate specific Ag Prostate Ca prostate 13. Lipase Pancreas Pancreatitis 14. Trypsin Pancreas Cystic fibrosis

145. ISOENZYMES 7/31/2020 168

146. DEFINITION Physically distinct forms of the same enzyme catalyzing same chemical reaction but differ in their physical & chemical properties.

147. DIAGNOSTICALLY IMPORTANT ISOENZYMES Creatine phosphokinase (CK/ CPK) Lactate dehydrogenase (LDH) Alkaline phosphatase (ALP)

148. CREATINE PHOSPHOKINASE Cytosolic enzyme Dimeric – M chain & B chain CK-1 = BB = Brain CK-2 = MB =Cardiac CK-3 = MM =Muscle

149. ISOENZYME EP MOBILITY TISSUE OF ORIGIN MEAN %AGE IN BLOOD CK – 3 =MM LEAST SKELETAL MUSCLE 94% CK – 2 =MB INTERMEDIATE HEART 5% CK – 1 =BB MAXIMUM BRAIN 1% CHARECTERISTICS OF CK ISOENZYMES

150. LACTATE DEHYDROGENASE (LDH) LD levels in tissues 500 times greater than serum levels ( cytosolic ) Liver - 145 U/gm Heart - 124 U/gm Kidney - 106 U/gm Skeletal muscle - 147 U/gm RBC - 36 U/gm of Hb

151. MW 134 Kda 4 polypeptide chains of 2 types – M chain & H chain 5 isoenzymes LD-1 : HHHH (H4) LD-2 : HHHM (H3M1) LD-3 : HHMM (H2M2) LD-4 : HMMM (H1M3) LD-5 : MMMM (M4)

152. STREPTOKINASE

153. Precautions for handelling specimen for enzyme estimation Digestion & absorption of carbohydrates, proteins & fats Factors influencing digestion & absorption Malabsorption syndrome 7/31/2020 176