Sept 5 bt202


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Sept 5 bt202

  1. 1. BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Sept 5, 2011
  2. 2. <ul><li>Enzyme kinetics </li></ul><ul><li>Catalytic mechanisms </li></ul><ul><li>Regulation of enzyme activity </li></ul><ul><li>Enzyme inhibition </li></ul>
  3. 3. <ul><li>Louis Pasteur (1850s): ferments, vitalism </li></ul><ul><li>Eduard Buchner (1897): fermentation is done by molecules </li></ul><ul><li>Frederick W. Kuhne: coined the name enzyme </li></ul><ul><li>James Sumner (1926): isolated urease and said enzymes are made up of proteins. </li></ul><ul><li>J.B.S. Haldane: weak interactions between enzyme and substrate are important for catalysis </li></ul>
  4. 4. Enzymes <ul><li>Definition; </li></ul><ul><li>Chemical reactions in cells require </li></ul><ul><li>specific catalysis. </li></ul><ul><li>Enzymes are proteins which perform </li></ul><ul><li>this function. </li></ul><ul><li>- 12kDa - 1,000kDa or more </li></ul><ul><li>- larger than their substrate </li></ul>
  5. 5. Enzymes <ul><li>Metabolite acted upon is called the enzyme ’s substrate. </li></ul><ul><li>Active site </li></ul><ul><li>- some residues involved in binding substrate </li></ul><ul><li>-others catalyze reaction </li></ul>
  6. 6. <ul><li>Cofactors </li></ul><ul><li>for some reactions, the amino acids are not powerful enough for catalysis. Enzymes overcome this by incorporate additional factors. </li></ul><ul><li>- metal ions as cofactors Zn 2+ , Fe 2+ , Cu 2+ </li></ul><ul><li>-coenzymes are organic cofactors </li></ul><ul><li>Prosthetic Group </li></ul><ul><li>Coenzyme or metal ion cofactor bound to enzyme either tightly or covalently. </li></ul><ul><li>- holoenzyme </li></ul><ul><li>-apoenzyme </li></ul>
  7. 7. Cofactor and Coenzymes
  8. 8. Properties of Enzymes <ul><li>Enzymes are excellent catalysts </li></ul><ul><li>- speeding up reactions 10 8 to 10 20 fold </li></ul><ul><li>Not used up during a reactions </li></ul><ul><li>Specificity for substrate </li></ul><ul><li>- absolute (eg., DNA polymerase) </li></ul><ul><li>- broad range (eg., synthesis of secondary metabolites) </li></ul><ul><li>Regulated- some enzymes can sense </li></ul><ul><li>metabolic signals. </li></ul>
  9. 9. Classification of Enzymes <ul><li>named and classified according to the substrate acted upon and the reaction catalyzed. </li></ul><ul><li>trivial names- end in –ase . (eg., urease, hexokinase. </li></ul><ul><li>based on a formal systemic catalog (IUB) with six major classifications. </li></ul>
  10. 11. Reaction Rates and the Transition State <ul><li>In order to react, the molecules involved </li></ul><ul><li>are distorted, strained or forced to have </li></ul><ul><li>an unlikely electronic arrangement. </li></ul><ul><li>That is the molecules must pass through a </li></ul><ul><li>high energy state. </li></ul><ul><li>This high energy state is called the transition state . </li></ul>
  11. 12. <ul><li>The energy required to achieve it is called </li></ul><ul><li>the activation energy for the reaction. </li></ul>The higher the free energy change for the transition barrier, the slower the reaction rate.
  12. 13. Enzymes lower energy barrier by forcing the reacting molecules through a different transition state.
  13. 14. Eg., C 12 H 22 O 11 + 12O 2 12CO 2 + 11H 2 O Reaction Intermediates Transient chemical species formed during a rxn Rate limiting step When many steps are involved the overall rate of the Reaction is determined by the step which has the Highest activation-energy
  14. 15. Relationship between reaction equilibrium and free energy Equilibrium constant is directly related to free energy Large negative free energy favors reaction
  15. 16. Rate of a reaction Unimolecular or first order reaction; V=k[S] Units are s -1 Rate for second order reaction; V= k[S 1 ][S 2 ] for transition state k = k T / h e - Δ G/RT Lower activation energy means faster rxn rates
  16. 17. How enzymes do it? Binding energy ( Δ G B ) Energy derived from enzyme substrate interaction. -transient covalent bond in the active site -non-covalent interactions to form ES
  17. 19. Role of binding energy in catalysis k = k T / h e - Δ G/RT Δ G + must be lowered by 5.7kJ/mol to accelerate first order reaction.
  18. 20. <ul><li>Specificity is derived from the formation of many weak interactions between the enzyme and its specific substrate. </li></ul><ul><li>Entropy reduction </li></ul>
  19. 21. <ul><li>Desolvation of substrate </li></ul><ul><li>ES complex </li></ul><ul><li>Enzyme undergoes conformation changes i.e. induced fit, postulated by Daniel Koshland (1958) </li></ul>
  20. 22. Modes of Enzymatic Enhancement of Rates <ul><li>Involve transient covalent interactions with a substrate or group transfer to or from a substrate </li></ul><ul><li>General acid-base catalysis </li></ul><ul><li>-good proton donors & acceptors positioned just right. </li></ul>
  21. 24. <ul><li>covalent catalysis </li></ul><ul><li>-unstable intermediate </li></ul><ul><li>metal ion catalysis </li></ul><ul><li>-electron donor or acceptor </li></ul>
  22. 25. <ul><li>At low concentrations of [S] V o increases almost linearly </li></ul><ul><li>At higher [S] concentration V o increases by smaller amounts in response to increase in [S] </li></ul><ul><li>Finally increase in V o is negligible as [S] increases </li></ul>Enzyme kinetics
  23. 26. Steady state kinetics <ul><li>rate stops increasing or plateaus because the complex ES becomes filled at high [S] </li></ul><ul><li>Pre-steady state and steady state </li></ul><ul><li>Introduced by Briggs and Haldane </li></ul>
  24. 27. Michaelis-Menten Model <ul><li>Rate equation </li></ul>
  25. 28. Lineweaver-Burk equation Double reciprocal plot
  26. 29. Interpreting V max and K m Steady state kinetics is the means by which biochemists can compare and characterize The catalytic efficiencies of enzymes.
  27. 30. K cat it is the limiting rate of any enzyme catalyzed reaction. Eg. , K cat is first order rate constant has units s -1 . It is also called Turnover number ( the number of substrate molecules converted to product) .
  28. 31. <ul><li>Specificity constant compares the catalytic </li></ul><ul><li>efficiencies of different enzymes or the turnover </li></ul><ul><li>of different substrates by the same enzyme. </li></ul><ul><ul><ul><ul><ul><li>V o = k cat / K m [E t ][S] </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>-V 0 depends upon [Et] and [S] </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>-k cat /K m is a second order rate constant </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>-units M -1 s -1 </li></ul></ul></ul></ul></ul>
  29. 32. Reactions with more than one substrate
  30. 33. Learning check! <ul><li>In a simple enzyme catalyzed rxn., the sum of </li></ul><ul><li>which two chemical species is strictly constant? </li></ul><ul><li>[E] + [ES] </li></ul><ul><li>[E] + [P] </li></ul><ul><li>[S] + [P] </li></ul><ul><li>[ES] + [P] </li></ul><ul><li>2. For a simple enzymatic rxn., what is the value of </li></ul><ul><li>The initial velocity when S=5km? </li></ul><ul><li> 5 Vmax </li></ul><ul><li>5/2 Vmax </li></ul><ul><li>4/5 Vmax </li></ul><ul><li>5/6 Vmax </li></ul>
  31. 34. <ul><li>Match the following </li></ul><ul><li>Michaelis complex The velocity at t=0 </li></ul><ul><li>Michaelis constant Km Also known as turnover number </li></ul><ul><li>Initial velocity The enzyme substrate complex </li></ul><ul><li>Maximal velocity The velocity when the enzyme is saturated with substrate </li></ul><ul><li>Catalytic velocity The conc. of substrate @ which the velocity is half maximal </li></ul><ul><li>rxns. are enzyme reactions with two substrates. </li></ul><ul><li>Bisubstrate </li></ul><ul><li>In Lineweaver-Burk plot of a simple enzymatic rxn. , what is the value of the y-intercept @ 1/Vo axis? </li></ul><ul><li>Vmax </li></ul><ul><li>1/Vmax </li></ul><ul><li>Km </li></ul><ul><li>1/Km </li></ul>
  32. 35. Regulatory enzymes Exhibit increased or decreased catalytic activity in response to certain signals. -Reversible non-covalent modulation - allosteric modulators -small metabolites or cofactors -Reversible covalent modification -inhibition with separate regulatory protein -proteolytic cleavage
  33. 36. Allosteric enzymes -Homotropic -Heterotropic
  34. 37. Heterotropic Allosteric Modulation In multienzyme pathways the regulatory enzyme is inhibited by the end product. Eg., L-Threonine to L-Isoleucine in bacteria.
  35. 38. Kinetics of Allosteric enzymes -sigmoid curve is observed -substrate concentration is represented by [S] 0.5 or K 0.5 homotropic allosteric enzymes
  36. 39. -reflects cooperative interaction between protein molecules -small changes in conc. of modulator can be associated with large changes in activity
  37. 40. Heterotropic Allosteric enzymes -Activation may cause increased velocity for fixed K 0.5 -Negative modulator cause decreased velocity for fixed K 0.5
  38. 41. -increased V max with little change in substrate
  39. 42. Learning check! Match the following Competitive Inhibiton decreases Km and Vmax Uncompetitive inhibition decreases [E]T Mixed inhibition increases Km Inactivation decreases Vmax, while Km may increase or decrease Mixed inhibition is characterized by two dissociation constants for the inhibition. True False
  40. 43. Reversible covalent modification
  41. 45. How do the modulators act? -oxygen atom of phosphoryl group undergo H-bond formation -repulsion of neighboring residues with negative charges phosphatase kinase
  42. 46. Glucose-6-phosphate ATP synthesis in muscles Free glucose in liver
  43. 48. -phosphorylation occurs in structural motif called consensus sequence -AA sequence is not the only factor which determines phosphorylation
  44. 49. Regulation by proteolytic cleavage -zymogen an inactive precursor is cleaved to form the active enzyme
  45. 50. -precursors are also called proprotein eg., collagen, fibrin, and thrombin
  46. 52. Enzyme inhibitors Interfere with catalysis, slowing or halting enzymatic reactions. -Reversible Inhibition -competitive Since it is reversible so increasing the concentration of substrate remove inhibition.
  47. 53. Uncompetitive inhibition Lower the measured V max Apparent K m also decreases
  48. 54. Mixed Inhibition
  49. 55. Irreversible Inhibition DIFP Diisopropylfluorophosphate Suicide inactivators such compounds are inactive untill they bind active site of a specific enzyme.