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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 63 101 Catalytic 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 curve ENZYME INHIBITOR ACTIVATOR ALA synthase heme phosphofructokinase ATP, citrate AMP Aspartate 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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