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Enzymes

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Enzymes

  1. 1. ENZYMES-Keshava Pavan K
  2. 2. • Prosthetic group – tightly bound coenzymes – FAD, FMN, Biotin• Cosubstrates – loosely bound coenzymes – NAD+, NADP+
  3. 3. • Coenzymes transferring H : – FAD, FMN, NAD, NADP- water soluble vitamins – Tetrahydrobiopterin – Lipoic acid – Coenzyme Q
  4. 4. • Coenzymes transferring groups other than H – Biotin (CO2) – Pyridoxal phosphate (amino) – CoA (acyl) – Tetrahydro folic acid (1 C groups) – Methyl cobalamine (CH3) – ATP (PO4) – S Adenosyl methionine (CH3) – UDP (glucose/galactose)• Last three do not belong to Vitamin B complex
  5. 5. • Activators are metal ions – tightly bound- metalloenzymes – loosely bound- metal activated enzymes• Stabilise proper conformation• Cu2+ for tyrosinase, Mg2+ for kinases• Fe, Cu in oxidation-reduction reactions• Pyruvate dehydrogenase complex requires 5 activators: Mg2+, NAD+, FAD+, TPP, CoA• Xanthine oxidase requires FAD, Mo, Fe
  6. 6. • Multifunctional enzymes: same enzyme molecule having different functions for different parts of that enzyme molecule – fatty acid synthase complex• Multienzyme complex: many enzymes clustered together having common function
  7. 7. • Active site of lysozyme: Glu Asp Trp Trp Asp 35 52 62 63 101Catalytic site substrate binding site• Hexokinase- 0.02 mmol L-1 : always active• Glucokinase- 10 mmol L-1 : only when glucose concentration is high, only in liver
  8. 8. CLINICAL APPLICATIONS OF COMPETITIVE INHIBITION• Sulphonamides(NH2-C6H5-SO2-NH2) resemble PABA (NH2-C6H5-COOH), hence inhibits it• Anticancer drugs like methotrexate, aminopterin inhibit dihydrofolate reductase• Alcohol dehydrogenase catalyses conversion of methyl alcohol to formaldehyde. Ethyl alcohol inhibits it. Therefore ethyl alcohol is used during methyl alcohol poisoning.
  9. 9. • Allopurinol is used to treat gout as it inhibits xanthine oxidase that converts hypoxanthine to uric acid• Dicoumarol is an anticoagulantas it inhibits Vit K needed for coagulation• Lovastatin inhibits HMG-CoA reductase, hence used to reduce cholesterol level in blood
  10. 10. NONCOMPETITIVE INHIBITORS• Cyanide inhibits cytochrome oxidase• Iodoacetate inhibits enzynes having –SH (sulfhydryl) group at active site like glyceraldehyde-3-phosphate dehydrogenase• Fluoride inhibits enolase (binding with Mg 2+ or Mn2+)• Di-isopropyl fluorophosphate (DFP) inhibits enzymes having serine at active site like chymotrypsin, acetylcholine esterase
  11. 11. SUICIDE INHIBITION• Irreversible• Mechanism based inhibition• Allopurinol becomes alloxanthine which is a more potent inhibitor• 5-fluoro uracil becomes fluoro deoxy uridylate which binds to the enzyme thymidylate synthetase and inhibits it
  12. 12. ALLOSTERIC MODULATION• Regulation of enzyme activity in the body• Positive & negative modulators• Not a substrate analog• Reversible• Allosteric site other than active site• Brings about conformational change• Mostly oligomeric enzymes – Aspartate transcarbamoylase (6 polypeptide chains) – Pyruvate kinase (4 polypeptide chains)
  13. 13. • Have sigmoidal curveENZYME INHIBITOR ACTIVATORALA synthase hemephosphofructokinase ATP, citrate AMPAspartate transcarbamoylase CTP ATP
  14. 14. UNCOMPETITIVE INHIBITION• Cannot bind with free enzyme, only to E-S complex inhibitor• 1/V 1/S• Eg,. Inhibition of placental alkaline phosphatase (Regan isoenzyme) by phenyl alanine
  15. 15. REGULATION OF ENZYME ACTIVITY• Induction (depression) and repression – Some are constitutive enzymes like hexokinase – Others are inducible like glucokinase• Allosteric modulation• Covalent modulation – Zymogen activation, phosphorylation and dephosphorylation• Compartmentalisation of pathways• Degradation• isoenzymes
  16. 16. DIAGNOSTIC ENZYMES• Aspartate transaminase – Normal 2 – 40 IU/L – Increases during MI & liver diseases• Alanine transaminase – Normal 0 – 45 IU/L – Very high during liver diseases – Moderately high during MI
  17. 17. • Alkaline phosphatase – Hydrolysis of phosphate esters – Optimum pH 9.5-10 – 25-100 IU/L – Increases in liver & bone diseases – Very high in cholestatic/obstructive jaundice• Acid phosphatase (prostatic fraction) – Optimum pH 4.5 – Increases in bone diseases & prostate cancers – Prostatic specific antigen-protease • 5mg/L • 4 to 10mg/L benign; >10 mg/L cancerous
  18. 18. • 5’ nucleotidase – Normal 2-10 IU/L – High in liver diseases, very high in cholestasis• γ- glutamyl transpeptidase – Normal 10-30 IU/L – High in liver diseases, very high in alcoholics• Cholinesterase – Normal 2-121 IU/L – Inhibited by organophosphorous pesticides• Pseudocholinesterase – Decreases in liver diseases
  19. 19. • Amylase – Normal 50-120 IU/L – Very high in acute pancreatitis – Moderately high in chronic pancreatitis & mumps• Lipase – Normal 0.2-1.5 IU/L – Diagnostics same as above; also high in cholestasis
  20. 20. THERAPEUTIC ENZYMES• Streptokinase – To dissolve intravascular clots• Asparginase – To treat leukemia• Pancreatin – Lipase, trypsin – To treat pancreatic insufficiency• Collagenase – To remove scar tissue
  21. 21. ANALYTICAL ENZYMES• Glucose oxidase, peroxidase – Estimation of glucose• Cholesterol oxidase – Estimation of cholesterol• Horse- raddish peroxidase- ELISA• Taq polymerase- PCR• Restriction endonuclease- rDNA technology & DNA fingerprinting
  22. 22. RIBOZYMES• snRNA/small nuclear RNA – Splicing• Peptidyl transferase – translation

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