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Enzymology BIOCHEMISTRY REVISION NOTES

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Enzymology BIOCHEMISTRY REVISION NOTES

  1. 1. Enzymology
  2. 2. • Biocatalyst • Usually proteins except ribozyme (RNA particles with catalytic activity ) • With out being changed themselves
  3. 3. Enzyme activity Unit of enzyme activity • Amount causing transformation of 1 um of substrate /min at 25 *C • Expressed as mole of substrate utilised or mole of product formed Specific activity • No of protein units /mg of protein • Measure of enzyme purity • Higher the enzyme purity  higher the specific activity Turn over number • Number of substrate molecules transformed per unit time by a single enzyme molecule (when enzyme concentration alone is limiting factor) • Highest turnover  catalase (fastest enzyme) followed by carbonic anhydrase • Lowest turnover  lysozyme (slowest enzyme)
  4. 4. Some enzymes are produced as proenzymes • Inactive precursors  zymogens or proenzymes • Proelastase • Pepsinogen
  5. 5. Co enzyme • organic molecule required by enzyme Co factor • Inorganic molecule required by enzyme
  6. 6. Nomenclature of enzyme • EC stands for enzyme commission, and • the first digit stands for the class name (transferase), • the second digit stands for the subclass • 3rd digit sub sub class • 4th digit  individual enzyme
  7. 7. Isomerases • Decarboxylases • Aldolases
  8. 8. Ligases • Synthetase • Carboxylases • Ligases
  9. 9. Co enzymes • Water soluble vitamins • Can function either as co substrate or prosthetic group
  10. 10. Sulfite oxidase
  11. 11. Molybdenum • Xanthine oxidase • Sulfite oxidase
  12. 12. Mechanism of enzyme action
  13. 13. • Most enzyme – substrate combinations are mostly d/t weak non covalent modification like hydrogen bond hydrophobic interactions & van der waal forces • Increase the rate of biochemical reaction • Lowering the magnitude of the activation energy barrier • decreasining free energy of activation
  14. 14. Lowering of activation energy
  15. 15. Michaelis menten theory Enzyme combines with a substrate to form a transient enzyme substrate complex Which break in to enzyme products
  16. 16. Temperature & enzyme activity Rate of enzyme activity increases with increase in temperature d/t increase in kinetic energy
  17. 17. Enzyme & pH
  18. 18. • Rate of reaction increases directly with increase in enzyme cncentration
  19. 19. Hyperbolic curve is obtained
  20. 20. Michaelis menten equation • Reaction velocity varies with substrate concentration • V0initial velocity • Vmax  maximum velocity • Km michaelis menten constant • [S] substrate concentration
  21. 21. Km (michaelis menten constant) • Substrate concentration at which reaction rate is half maximum • Constant for each enzyme • Reflects binding affinity of the enzyme for its substrate • High enzyme substrate affinity implies a low Km value • Low affinity implies high Km • Natural substrate has lowest km • key enzyme has highest km
  22. 22. Lineweaver burk plot
  23. 23. Lineweaver burk plot is also known as double reciprocal plot
  24. 24. Fischers lock and key theory
  25. 25. Koshlands induced fit theory
  26. 26. Enzyme inhibition
  27. 27. Enzyme inhibition • Competitive inhibition • Noncompetitive inhibition • Suicide inhibition • Allosteric inhibition • Feedback inhibition
  28. 28. Competitive inhibition • Inhibitor is structural analog of substrate • Binds to same site as substrate • Km increases • Reversible • excess substrate abolishes inhibition • Vmax remains same • Km increase
  29. 29. Vmax remains same Km increases (decreased affinity to substrate)
  30. 30. • Competitive inhibition • Eg • Statin on HMG coA reductase • MTX on DHFR • Dicumarol in vitamin K epoxide • Succinate dehydrogenase by malonate
  31. 31. • Effect on Vmax: • The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor . • Effect on Km: • A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve 1⁄2Vmax.
  32. 32. Non competitive inhibition • Can be reversible or irreversible • Mostly irreversible • Inhibitor have no structural similarity to substrate • Bind to site other than substrate binding site • Affinity to substrate same  Km remains same • Excess substrate donot abolish inhibition • Vmax decreases • Less enzyme activity
  33. 33. • 1. Effect on Vmax: • Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, noncompetitive inhibitors decrease the apparent Vmax of the reaction. • 2. Effect on Km: • Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor.
  34. 34. Non competitive inhibition • Cyanide on cytochrome oxidase
  35. 35. Vmax remains same Km increases (decreased affinity to substrate)
  36. 36. Uncompetitive inhibitor • Bind only to enzyme substrate complex  ESI complex • Decrease in both Vmax • Km reduced • Phenylalanine & placental ALP
  37. 37. Suicide inhibition • Mechanism based inactivation • Inhibitors are unreactive until they are converted by enzyme in to active product • Which in turn inhibits the enzyme • Eg aspirin COX • Allopurinol xanthine oxidase
  38. 38. Suicide inhibition by aspirin
  39. 39. Feed back inhibition • End product inhibition
  40. 40. Regulation of enzymes
  41. 41. • Regulation of enzyme quality • Allosteric regulation • Covalent modification • Regualtion of enzyme quality • Control of enzyme synthesis  induction & repression • Control of enzyme degradation
  42. 42. Allosteric regulation • Allosteric enzymes have a separate site where a modifier binds other site for binding of substrates • Can be • Allosteric activator • Allosteric inhibitor
  43. 43. • Allosteric enzymes have a quarternary structure & made up of sub units
  44. 44. Cooperative binding  sigmoid shaped curve
  45. 45. Hills equation describes the allosteric modification
  46. 46. Covalent modification
  47. 47. Covalent modification • Reversible • Phosphorylation /dephophorylation • Methylation • Adenylation • Acetylation • Irreversible • Partial proteolysis /zymogen activation
  48. 48. Phosphorylation  most common covalent modification Enzymes active in phosphorylated state • Glycogen phosphorylase • Key enzymes of gluconeogenesis Enzymes active in dephosphorylated sate • Glycogen synthase • Enzymes of glycolysis
  49. 49. Caspaces
  50. 50. • Catalase • Highest turn over • Fastest acting enzyme
  51. 51. • Serine Proteases • They are enzymes with a serine residue at the active site and most of the proteolytic enzymes belong to this group, e.g. trypsin, chymotrypsin, clotting factors
  52. 52. Clinical enzymology
  53. 53. • Nonfunctional plasma enzymes • Cell derived enzymes • Released in to plasma by autolysis (necrosis) or increased permeability of cells
  54. 54. • Functional enzyme in plasma • Clotting factors in plasma
  55. 55. Enzymes in Heart • CK • LDH • AST
  56. 56. CK MB • First enzyme to elevate • Rises in 3- 8 hrs • Remain elevated for 3 days • Early diagnosis of MI
  57. 57. CK is a dimer made up of 2 subunits B & M subunits
  58. 58. Fetal reversion • Damaged skeletal muscle may contain more CK-MB owing to phenomenon of fetal reversion, thus serum CK-MB isoenzyme may increase in such conditions. • In c/c muscle disorders
  59. 59. LDH • Tetramer made of H & M subunits • 5 isoenzymes • Normally in blood LDH2 > LDH1 • But in MI  LDH 1 > LDH2 } FLIPPED PATTERN
  60. 60. • LDH • Elevated after 12- 18 hrs (last enzyme to elevate) • Peak value in 3 days • Remain elevated for 14 days late diagnosis of MI
  61. 61. LDH
  62. 62. • LDH 2 is elevated in anemia
  63. 63. • LDH may also be elevated in haemolysis
  64. 64. AST • Elevated in MI & hepatocellular damage • Non specific • Elevated after days & lasts for less duration • Not used in diagnosis
  65. 65. Pro BNP • The best marker of ventricular dysfunction is pro-BNP.
  66. 66. Ischemia modifed albumin (IMA) • Myocardial ischemia alters the N-terminus of albumin reducing the ability of cobalt to bind to albumin. • Rises with in 6 – 10 minutes • Low specificity (ischemia in any organ) • negative value is highly useful, as it rules out the possibility of MI
  67. 67. Myocardial ischemia alters the N-terminus of albumin reducing the ability of cobalt to bind to albumin
  68. 68. Myoglobin • Low specificity
  69. 69. • Troponin I • is released into the blood within 4 hours after the onset of symptoms of myocardial ischemia; peaks at 14–24 hours and remains elevated for 3–5 days postinfarction • Troponin T (TnT) • increases within 6 hours of myocardial infarction, peaks at 72 hours and then remains elevated up to 10–14 days
  70. 70. Troponin • Skeletal muscle cardiac muscle • Not in smooth muscle • Any time marker for MI
  71. 71. H -FABP • Heart type fatty acid binding protein • Used as predictor of death or MI @ 1 yr in ACS • H FABP – ve  low mortality • H FABP +ve  high mortality
  72. 72. Enzymes in liver disease
  73. 73. Enzymes in liver disease • Hepatocellular damge • ALT • AST • Obstructive • ALP • Gamma glutamyl transferase • 5 nucleotidase
  74. 74. AST / ALT • AST /ALT <1 :viral hepatitis • AST /ALT 1-2 : cirrhosis • AST /ALT >2 : alcoholic hepatitis or HCC
  75. 75. Isoenzymes of ALP • Alpha 1 ALP- • epithelial cells of biliary canaliculi • increased in obstructive jaundice • Alpha 2 heat labile ALP- • hepatic cells • Moderate elevation in jaundice • Alpha 2 heat stable ALP placental • -not destroyed at 65˚C inhibited by phenylalanine • Pre beta ALP – bone,heat labile • Gamma ALP – • intestinal cells inhibited by phenylalanine • Elevated in ulcerative colitis • Leukocyte alkaline phosphatase • –decreased in CML increase in lymphoma
  76. 76. • ATYPICAL ISOENZYMES • Regan isoenzyme-heat stable,inhibited by L-phenylalanine • a/w ca liver lung GIT • Also increased in smokers • Nagao isoenzyme- variant of regan inhibited by L-leucine • In pleural malignancy
  77. 77. 5 ‘ nucleotidase • 2-17 U/L • Elevated in obstructive ds
  78. 78. GGT (gamma glutamyl transferase ) • Very sensitive for alcoholic liver ds • Present on membrane surface
  79. 79. Pancreatic ds • S amylase • Non specific • Increased in all cases of a/c abdomen • Small protein excreted in urine  elevated in renal failure • S lipase • Specific
  80. 80. Prostate • Acid phosphatase • PSA
  81. 81. Acid phosphatase • Secreted by prostate cells, RBC, platelets and WBC. • prostate iso-enzyme is inactivated by tartaric acid • Erythrocytic form is inhibited by cupric ions • increased in prostate cancer and highly elevated in bone metastasis of prostate cancer • tartrate labile iso-enzyme is elevated. • This assay is very helpful in follow-up of treatment of prostate cancers. Tartrate resistant acid phosphatase • Elevated in osteoclastoma • Osteodystrophy • Metabolic bone ds
  82. 82. PROSTATE SPECIFIC ANTIGEN (PSA) • serine protease • Normal value is 1–5 mg/L. • It is very specifc for prostate activity. • Values above 10 mg/L is indicative of prostate cancer
  83. 83. Anti tranglutaminase activity • In celiac ds • Sensitve & specific
  84. 84. Abzyme • Ab with enzymatic activity

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