Enzymes

1. Enzymes

2. Biologic Catalyst • Enzymes are neither consumed nor produce during the course of a RXN • They greatly enhances the rate of reaction that would proceed much slower in their absence -Do not cause the reaction to take place, but alter the rate, not the equilibrium constant of reaction they catalyzes

3. Enzyme Nomenclature • All enzymes has two names: • Recommended /Trivial Name End in the suffix “ase” Describe the substrate or description of the action perform b) Systematic Name - 6 classes each with subgroups -Classification number

5. Active site • A part of the enzyme that participate in substrate binding and catalysis • Binding of substrate causes a conformational change in that allow catalysis Theories: • Lock and Key • Induce Fit

6. Characteristics of enzyme active sites Catalytic site Where the reaction actually occurs Binding site Area that holds substrate in proper place Enzymes uses weak, non-covalent interactions to hold the substrate in place based on R groups of amino acids Shape is complementary to the substrate and determines the specificity of the enzyme Sites are pocket or clefts on the enzyme

8. Properties of enzymes • 1) Catalytic Efficient • 2) Highly Specific • 3) Capacity for regulation • 4) Location within cell • 5) High reaction rates • 6) Holoenzymes

9. Holoenzyme Enzyme with Non protein content • Apoenzyme Inactive due to absent of Non Protein content

12. Enzymes provide an alternate energetically favorable reaction pathway

13. Factors that influence enzyme activity • Effect of pH on enzyme activity

14. Exceeding the optimum pH and temperature reduces the enzyme activity -Optimum temperature is usually 25-40 0 c ,for pH, a lot of enzymes work best near pH 7

16. Leveling of the graph suggest the active site is saturated with substrate

17. Formation of Enzyme Substrate Complex

18. Formation of the transition state

19. Formation of the Enzyme-product complex

20. Formation of the product

21. Michaelis - Menten Kinetic Model • E + S ↔ ES → E + P • Describes how reaction velocity varies with Substrate concentration • Vmax = k2 [E]

22. Michaelis-Menten Kinetics Km is equal to the concentration of substrate required to attain half maximal velocity for any given reaction

23. Understanding Km Conclusion about M-M • The "kinetic activator constant" • Km is a constant derived from rate constants • Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S • Small Km means tight binding; high Km means weak binding

24. The catalytic efficiency • When [S] < Km, velocity of the reaction is approx. equal to the substrate concentration • -First order with respect to the substrate • When [S] ˃ Km, velocity of the reaction is constant and independent of substrate concentration • -Zero order with respect to the substrate

26. Lineweaver –Burk Plot

27. Lineweaver-Burk Analysis • Lineweaver and Burk manipulated the MM equation by taking its reciprocal values generating a double reciprocal plot • Leads to a linear graph of the reciprocals of velocity and substrate concentration

28. Enzyme Inhibition • Inhibitors diminish the velocity of enzyme catalyzed reactions Many substances can inhibit enzyme activity: substrate analogues toxins drugs metal complexes Inhibition studies can provide: Information on metabolic pathways Insight on how drugs and toxins exert their effects Better understanding of enzyme reaction mechanisms

29. Inhibitors Irreversible Forms a covalent or very noncovalent bonds Reversible Forms weak, noncovalent bonds that readily dissociate from an enzyme -competitive -noncompetitive -uncompetitive

30. Enzyme Inhibition - Summary • Competitive • Inhibitor binds reversibly at active site, inhibition is reversible as higher substrate competes for inhibitor, Vmax unchanged, Km increased • Noncompetitive • Inhibitor binds at site other than substrate, ESI cannot form product preventing the reaction from occurring, increased substrate does not compete, Km unchanged, Vmax decreased • Uncompetitive • Inhibitoronly binds to ES complex due to binding site becoming available only when substrate is bound, Km and Vmax decreased

31. Enzyme inhibition • Irreversible inhibitors • permanently inactivate enzymes • heavy metals (Hg2+, Pb2+, Cd2+) • aspirin acetylates • fluorouracil • organophosphates

33. EnzymeInhibition • Examples of competitive inhibitors: • methanol and ethylene glycol compete with ethanol for the binding sites to alcohol dehydrogenase • methotrexate competes with folic acid for dihydrofolate reductase • HMG Co A inhibitors

35. Enzyme inhibition • Examples of noncompetitive inhibitors: • physostigmine is a cholinesterase inhibitor used in the treatment of glaucoma • captopril is an ACE inhibitor used in treatment of hypertension • allopurinol is a xanthine oxidase inhibitor used to treat gout • Aspirin

37. Enzyme inhibition • Uncompetitive inhibitor • similar to a noncompetitive inhibitor but only • binds to the ES complex

40. Clinically Useful Competitive Inhibitors Drug Enzyme inhibited Clinical Use Allopurinol xanthine oxidase gout Dicoumarol Vit.K-epoxide reductase anti-coagulant Sulfonamide pteroid synthetase anti-biotic Methotrexate folic reductase anti-cancer 9-fluorouracil thymidilate synthase anti-cancer Azaserine phosphoribosyl anti-cancer amidotransferase Acyclovir DNA polymerase anti-viral Lovastatin HMG-CoA reductase cholesterol

42. Other methods of enzyme regulation Feedback inhibition (heterotropic effector) A type of allosteric effect where the product acts as the effector molecule Proenzymes/Zymogens Inactive forms of an enzyme Cell activates them on demand using another enzyme(proteases). A portion of the protein is removed. Covalent Modification – addition of phosphate or removal of phosphate from serine, threonine, tyrosine Induction and Repression of enzyme synthesis

43. Enzymes in the clinic • Injury or death of tissues can cause the release of tissue-specific enzymes into the bloodstream. • Thus, elevated enzyme levels are often indicators of tissue problems, and are used in the diagnosis of disease.

44. Enzymes of Clinical Interest • Acid phosphatase (ACP) • Catalyzes removal of phosphate groups at acid pH. • Found in prostate, bone, liver, spleen, kidney,  RBCs, and platelets. • Primarily used to diagnose prostate cancer. • Normal value: 2.5-11.7 U/L (M); 0.3-9.2 U/L (F)

45. Enzymes of Clinical Interest • Alanine aminotransferase (ALT) (AKA SGPT) • Widely distributed, but high concentrations  are found in the liver. • Useful in diagnosis of liver disorders. • Normal: 6.0-21 U/L

46. Enzymes of Clinical Interest • Amylase (AMS) • Catalyzes the digestion of starch. • Found in pancreas and salivary glands. • Used to diagnose pancreatitis. • Also elevated in mumps (infects salivary glands) • Normal: 96-290 U/L (serum); 160-2000 U/L(urine)

47. Enzymes of Clinical Interest • Alkaline phosphatase (ALP) • Removes phosphate groups at alkaline pH. • Widely distributed, high concentrations in intestines, liver, bone, spleen, placenta and kidney. • Useful in diagnosis of hepatobiliary and bone disorders. • Also elevated during healing of fractures. • Normal: 25-90 U/L

48. Enzymes of Clinical Interest • Aspartate aminotransferase (AST) (AKA SGOT) • This enzyme is widely distributed in the body: • Cardiac and skeletal muscle, and liver. •  Useful in the diagnosis of MI, liver disorders,  and muscle damage. • Normal : 7.0-20 U/L

49. Lactate Dehydrogenase (LDH) electrophoretic subunits mobility tissue of origin at pH 8.6 LDH-1 H4 fastest heart LDH-2 H3M1 faster RBC LDH-3 H2M2 fast brain LDH-4 H1M3 slow liver LDH-5 M4 slowest skeletal muscle

50. Creatine Kinase (CK) electrophoretic subunitsmobility tissue of origin at pH 8.6 CK3 MM least skeletal muscle CK2 MB int heart CK1 BB max brain CK Creatine-phosphate myofibrils (mitochondria) muscle contraction