enzymes kinetics inhibition regulation n.
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  1. ENZYMES KINETICS, INHIBITION, REGULATION Muhammad Jawad Hassan Assistant Professor Biochemistry

  2. Michaelis-Menten kinetics V0 varies with [S] Vmax approached asymptotically V0 is moles of product formed per sec. when [P] is low (close to zero time) E + SESE + P Michaelis-Menten Model V0 = Vmax x[S]/([S] + Km) Michaelis-Menten Equation

  3. Steady-state & pre-steady-state conditions At pre-steady-state, [P] is low (close to zero time), hence, V0 for initial reaction velocity At equilibrium, no net change of [S] & [P] or of [ES] & [E] At pre-steady state, we can ignore the back reactions

  4. Michaelis-Menten kinetics (summary) Enzyme kinetics (Michaelis-Menten Graph) : At fixed concentration of enzyme, V0is almost linearly proportional to [S] when [S] is small,but is nearly independent of [S] when [S] is large k1 k2 Proposed Model: E + S  ES  E + P ES complex is a necessary intermediate Objective: find an expression that relates rate of catalysis to the concentrations of S & E, and the rates of individual steps

  5. Michaelis-Menten kinetics (summary) Start with: V0 = k2[ES],and derive, V0 = Vmax x[S]/([S] + Km) At low [S] ([S] < Km), V0 = (Vmax/Km)[S] At high [S] ([S] > Km), V0 = Vmax When [S] = Km, V0 = Vmax/2. Thus, Km = substrate concentration at which the reaction rate (V0) is half max.

  6. Range of Km values Km provides approximation of [S] in vivo for many enzymes

  7. Lineweaver-Burk plot (double-reciprocal)

  8. Allosteric enzyme kinetics Sigmoidal dependence of V0 on [S], not Michaelis-Menten Enzymes have multiple subunits and multiple active sites Substrate binding may be cooperative

  9. Enzyme inhibition

  10. A competitive inhibitor

  11. Methotrexate A competitive inhibitor of dihydrofolate reductase - role in purine & pyrimidine biosynthesis Used to treat cancer

  12. Kinetics of competitive inhibitor Increase [S] to overcome inhibition Vmax attainable, Km is increased Ki = dissociation constant for inhibitor

  13. Competitive inhibitor Vmax unaltered,Km increased

  14. Kinetics of non-competitive inhibitor Increasing [S] cannot overcome inhibition Less E available, Vmax is lower, Km remains the same for available E

  15. Noncompetitive inhibitor Km unaltered,Vmax decreased

  16. Enzyme inhibition by DIPF Group - specific reagents react with R groups of amino acids diisopropylphosphofluoridate DIPF (nerve gas) reacts with Ser in acetylcholinesterase

  17. Affinity inhibitor: covalent modification

  18. Catalytic strategies commonly employed • Covalent catalysis.The active site contains a reactive group, usually a nucleophile that becomes temporarily covalently modified in the course of catalysis • 2. General acid-base catalysis.A chemical reaction is catalyzed by an acid or a base. The acid is often the proton and the base is often a hydroxyl ion. A molecule other than H2O may play the role of a proton donor or acceptor.

  19. 3. Metal ion catalysis.Metal ion can function in several ways; • can serve as an electrophile, stabilizing a negative charge on a reaction intermediate. • can generate a nucleophile by increasing the acidity of a nearby molecule, such as H2O in the hydration of CO2 by carbonic anhydrase. • can bind to substrate, increasing the number of interactions with the enzyme. 4. Catalysis by approximation.Bringing two substrates together along a single binding surface on an enzyme

  20. Enzyme specificity: chymotrypsin Cleaves proteins on carboxyl side of aromatic, or large hydrophobic amino acid Bonds cleaved, indicated in red The enzyme needs to generate a powerful nucleophile to cleave the bond

  21. A highly reactive serine (#195) in chymotrypsin 27 other serines not reactive to DIPF, Ser 195 is a powerful nucleophile DIPF: di-isopropylphosphofluoridate,only reacts with Ser 195

  22. Covalent catalysis Hydrolysis in two stages Deacylation to regenerate free enzyme Acylation to form acyl-enzyme intermediate Ser 195 OH group attacks the carbonyl group Acyl-enzyme intermediate is hydrolysed

  23. Chymotrypsin in 3D 3 chains; orange, blue, & green Catalytic triad of residues, including Ser 195 2 interstrand, & 2 intrastrand disulfide bonds See Structural Insights Synthesized as chymotrypsinogen Proteolytic cleavage to 3 chains

  24. The catalytic triad (constellation of residues) Ser 195 converted into a potent nucleophile, an alkoxide ion Asp 102 orients His 57 Imidazole N as base catalyst, accepts H ion, positions & polarizes Ser H ion withdrawal from Ser 195 generates alkoxide ion

  25. Regulatory Strategies: Enzymes & Hemoglobin Allosteric control. Proteins contain distinct regulatory sites and multiple functional sites.Binding of regulatory molecules triggers conformational changes that affect the active sites. Display cooperativity: small [S] changes - major activity changes.Information transducers: signal changes activity or information shared by sites 2. Multiple forms of enzymes (isozymes). Used at distinct locations or times.Differ slightly in structure, in Km & Vmax values, and in regulatory properties 3. Reversible covalent modification. Activities altered by covalent attachment of modifying group, mostly a phosphoryl group 4. Protleolytic activation. Irreversible conversion of an inactive form (zymogen) to an active enzyme

  26. Aspartate transcarbamoylase reaction Committed step in pyrimidine synthesis:inhibited by end product CTP

  27. CTP inhibits ATCase

  28. CTP stabilizes the T state CTP binds to regulatory subunits

  29. R and T states in equilibrium

  30. ATCase displays sigmoidal kinetics Substrate binding to one active site converts enzyme to R state increasing their activity: active sites show cooperativity

  31. Basis of sigmoidal curve R & T states equivalent to 2 enzymes with different Kms Cooperativity

  32. Effect of CTP on ATCase kinetics CTP stabilizes the T state, curve shifts to right

  33. Effect of ATP on ATCase kinetics ATP, allosteric activator, stabilizes R state, curve shifts to left

  34. Oxygen delivery by hemoglobin, cooperativity enhanced 98 - 32 = 66% 63 - 25 = 38% Cooperativity enhances delivery 1.7 fold Partial pressure of oxygen

  35. Heme group structure 4 linked pyrrole rings form a tetrapyrrole ringwith a central iron atom. side chains attached

  36. Position of iron in deoxyhemoglobin Iron slightly outside porphyrin plane His (imidazole ring) binds 5th coordination site 6th site for O2 binding

  37. O2 binding,conformational change Iron moves into plane, his is pulled along

  38. Quaternary structure of hemoglobin Pair of identical alpha-beta dimers

  39. Transition from T-to-R state in hemoglobin Interface most affected As O2 binds, top pair rotate 15o with respect to bottom pair

  40. Oxygen affinity of fetalvmaternal red blood cells Fetal Hgl does not bind 2,3-BPG, higher O2 affinity Fetal hemoglobin tetramer has 2 alpha & 2 gama chains, Gene duplication

  41. Isozymes of lactate dehydrogenase: glucose metabolism Rat heart LDH isozyme profile changes with development H(heart) isozyme (chain)= square,M(muscle) isozyme = circle

  42. Tissue content of LDH Functional LDH is tetrameric, with different combinations of subunits possible. H4 (heart) has higher affinity for substrates than does M4 isozyme, different allosteric inhibition by pyruvate H4 H3M H2M2 HM3 M4 Some isozymes in blood indicative of tissue damage, used for clinical diagnosis Increase in serum levels of H4 relative to H3M, indicative of myocardial infraction (heart attack)

  43. Examples of covalent modification

  44. Phosphorylation widely used for regulation Gamma phosphoryl group

  45. Some known protein kinases

  46. Protein phosphotases Reverse the effects of kinases, catalyze hydrolytic removal of phosphoryl groups attached to proteins

  47. Activation by proteolytic cleavage

  48. Secretion of zymogens by acinar cell of pancreas Pancreas, one of the most active organs in synthesizing & secreting proteins Acinar cell stimulated by hormonal signal or nerve impulse, granule content released into duct to duodenum

  49. Proteolytic activation of chymotrypsinogen Active enzyme generated by cleavage of a single specific peptide bond 3 chains linked by 2 interchain disulfide bonds, (A-B & B-C)

  50. Conformations of chymotrypsinogen & chymotrypsin Electrostatic interaction between Asp 194 carboxylate & Ile 16 -amino group possible only in chymotrypsin, essential for activity