1. Copyright (c) by W. H. Freeman and Company LECTURE No.4 Enzymes: I] Catalytic Strategies (Ch.9) II] Regulatory Strategies (Ch.10)

2. Copyright (c) by W. H. Freeman and Company A few basic catalytic principles used by many enzymes zCovalent catalysis: transient covalent bond between enzyme and substrate zGeneral acid-base catalysis: other molecule than water gives/accept protons (Histidine) zMetal ion catalysis: several strategies possible zCatalysis by approximation: bringing substrates in proximity

3. Copyright (c) by W. H. Freeman and Company I] Catalytic strategies Covalent catalysis and General acid-base catalysis: the example of Chymotrypsin, a protease

4. Copyright (c) by W. H. Freeman and Company Chymotrypsin cleaves peptides ”after” non-polar bulky residues

5. Copyright (c) by W. H. Freeman and Company Chymotrypsin facilitates nucleophylic attack zAmide bond hydrolysis is thermodynamically favored but very slow zCarbon in carbonyl group resistant to nucleophilic attack: partial double-bond with N & planar geometry

6. Copyright (c) by W. H. Freeman and Company An unusually reactive Serine in Chymotrypsin, amongst 28

7. Copyright (c) by W. H. Freeman and Company Chromogenic substrate analogues to measure activity

8. Copyright (c) by W. H. Freeman and Company Kinetics of chymotrypsin

9. Copyright (c) by W. H. Freeman and Company A covalent ES complex to explain the ”burst phase”

10. Copyright (c) by W. H. Freeman and Company Active-site SER in binding-site pocket of Chymotrypsin

11. Copyright (c) by W. H. Freeman and Company Why is Ser195 so reactive? The catalytic triad Acid-base catalyst

12. Copyright (c) by W. H. Freeman and Company Catalytic cycle of Chymotrypsin

13. Copyright (c) by W. H. Freeman and Company Step 1: Substrate binding

14. Copyright (c) by W. H. Freeman and Company Step 2: Nucleophilic attack on carbonyl carbon

15. Copyright (c) by W. H. Freeman and Company Step 3: Acylation of Serine 195

16. Copyright (c) by W. H. Freeman and Company Step 4 & 5: Peptide (amine) leaves, Water comes in

17. Copyright (c) by W. H. Freeman and Company Step 6: Nucleophilic attack by water on the carbonyl carbon

18. Copyright (c) by W. H. Freeman and Company Step 7: Peptide (carbonyl) leaves, Serine 195 regenerated

19. Copyright (c) by W. H. Freeman and Company Stabilization of the tetrahedral intermediate

20. Copyright (c) by W. H. Freeman and Company Hydrophobic pocket of Chymotrypsin: S 1 pocket

21. Copyright (c) by W. H. Freeman and Company More complex, more specific hydrophobic pockets of other proteases Thrombin: Leu Val Pro Arg Gly Ser

22. Copyright (c) by W. H. Freeman and Company Chymotrypsin (red) and Trypsin (blue): structurally similar enzymes

23. Copyright (c) by W. H. Freeman and Company Structure of the S1 pockets explain substrate specificity

24. Copyright (c) by W. H. Freeman and Company Subtilisin active site pocket Subtilisin (Bacillus amyloliquefaciens) Chymotrypsin

25. Copyright (c) by W. H. Freeman and Company Structurally unrelated enzymes can develop identical strategies: convergent evolution Carboxypeptidase II from wheat

26. Copyright (c) by W. H. Freeman and Company Site-directed mutagenesis to unravel the function of catalytic residues K cat reduced by a factor 10 6

27. Copyright (c) by W. H. Freeman and Company Other proteases, other active sites...

28. Copyright (c) by W. H. Freeman and Company Alternative residues for a common strategy: nucleophilic attack.

29. Copyright (c) by W. H. Freeman and Company Structure of HIV protease II: an Aspartate protease

30. Copyright (c) by W. H. Freeman and Company HIV protease inhibitor that mirrors the twofold symmetry of the enzyme

31. Copyright (c) by W. H. Freeman and Company HIV protease – crixivan complex

32. Copyright (c) by W. H. Freeman and Company Structural rearrangement upon binding of crixivan (Chain A)

33. Copyright (c) by W. H. Freeman and Company I] Catalytic strategies Metal ion catalysis: the example of Carbonic Anhydrase II, an enzyme with prodigious catalytic velocity

34. Copyright (c) by W. H. Freeman and Company Hydration of CO 2 in the blood zNon catalyzed reaction happens at moderate pace: k 1 =0.15 s -1 (pH7.0, 37°C) zCarbonic anhydrase: K cat =600.000 s -1 zSpecial strategies to compensate for limiting factors (diffusion limits...)

35. Copyright (c) by W. H. Freeman and Company The active-site structure of human carbonic anhydrase II

36. Copyright (c) by W. H. Freeman and Company Carbonic anhydrase activity is strongly pH-dependent Active site group pK a close to 7.0

37. Copyright (c) by W. H. Freeman and Company When bound to Zn(II), pK a of water drops from 15.7 to 7.0

38. Copyright (c) by W. H. Freeman and Company Catalytic mechanism of carbonic anhydrase

39. Copyright (c) by W. H. Freeman and Company A synthetic analog mimicks carbonic anhydrase catalytic mechanism Water pK a =8.7 Hydration of CO 2, 100-fold at pH 9.2

40. Copyright (c) by W. H. Freeman and Company Kinetics of water deprotonation illustrates rate constants limitation zProton diffusion: k= 10 -11 M -1 s -1 zIn above reaction k -1 ≤ 10 11 M -1 s -1 at pH7.0, K=10 -7 M => k 1 ≤ 10 4 s -1 zProblem: k cat = 10 6 s -1 !

41. Copyright (c) by W. H. Freeman and Company Buffers displace the equilibrium constant zRate of proton loss is given by [B].k 1 ´ zBuffer diffusion: k= 10 9 M -1 s -1 zWith [B]=10 -3 M, [B]. k 1 ´= 10 -3 x10 9 =10 -6 s -1 zProblem: buffers not accessible to active site!

42. Copyright (c) by W. H. Freeman and Company Effect of buffer concentration on hydration of CO 2

43. Copyright (c) by W. H. Freeman and Company Histidine 64 shuttles protons from the active site to the buffer in solution

44. Copyright (c) by W. H. Freeman and Company  -carbonic anhydrases in archea: different structure but same function as carbonic anhydrase II from humans

45. Copyright (c) by W. H. Freeman and Company II] Regulatory Strategies zAllosteric control z(Isomerisation of enzymes: ”Isozymes”) z(Reversible covalent modifications) z(Proteolytic activation)

46. Copyright (c) by W. H. Freeman and Company II] Regulatory strategies Allosteric inhibition,”feedback” regulation: the case of Aspartate Transcarbamoylase (ATCase)

47. Copyright (c) by W. H. Freeman and Company Reaction catalyzed by ATCase ATCase

48. Copyright (c) by W. H. Freeman and Company Effect of cytidine triphosphate on ATCase activity (Gerhart & Pardee, 1962)

49. Copyright (c) by W. H. Freeman and Company Modification of cysteine residues induces changes in ATCase structure

50. Copyright (c) by W. H. Freeman and Company Changes in structure revealed by differencial sedimentation (ultracentrifugation) nativep-HMB treated Catalytic subunit Regulatory subunit

51. Copyright (c) by W. H. Freeman and Company Quaternary structure of ATCase (2C 3 + 3R 2 ) : ”Top-View” (x 2) Coordinated by 4x -SH

52. Copyright (c) by W. H. Freeman and Company Quaternary structure of ATCase (2C 3 + 3R 2 ) : ”Side-View”

53. Copyright (c) by W. H. Freeman and Company A bi-substrate analog to map the active- site residues

54. Copyright (c) by W. H. Freeman and Company X-ray crystallography reveals the substrate-binding site 3x2 active sites / enzyme

55. Copyright (c) by W. H. Freeman and Company Binding of PALA induces major conformational changes (Tense, lower affinity)(Relaxed, higher affinity)

56. Copyright (c) by W. H. Freeman and Company Molecular motion of the T-state to R- state transition

57. Copyright (c) by W. H. Freeman and Company Binding sites of cytidine triphosphate (CTP,effector) z 1x CTP binding-site per R unit z 50Å away from catalytic site z How does CTP inhibits activity?

58. Copyright (c) by W. H. Freeman and Company CTP induces a transition R  T state by a concerted mechanism [T]/[R]= 200

59. Copyright (c) by W. H. Freeman and Company Allosteric enzymes do not follow Michaelis-Menten kinetics Sigmoidal instead of hyperbolic

60. Copyright (c) by W. H. Freeman and Company Two additive Michaelis-Menten kinetics: T state + R state. Positive cooperativity! Sum of the two hyperbolic curves

61. Copyright (c) by W. H. Freeman and Company CTP an allosteric inhibitor of ATCase

62. Copyright (c) by W. H. Freeman and Company ATP an allosteric activator of ATCase

63. Copyright (c) by W. H. Freeman and Company Sequential models can account for allosteric effects zSeveral intermediate states can exist zBinding to one site influences affinity in neighboring site zNegative cooperativity

64. Copyright (c) by W. H. Freeman and Company II] Regulatory Strategies Hemoglobin: efficient O 2 transport by positive cooperativity

65. Copyright (c) by W. H. Freeman and Company Positive cooperativity enhances O 2 delivery by hemoglobin Hemoglobin increases by 1.7-fold the amount of oxygen delivered to the tissues

66. Copyright (c) by W. H. Freeman and Company Oxygen binding site in hemoglobin is a prosthetic group: the heme

67. Copyright (c) by W. H. Freeman and Company Non-planar porphyrin in deoxyhemoglobin

68. Copyright (c) by W. H. Freeman and Company Conformational change of the heme upon O 2 binding

69. Copyright (c) by W. H. Freeman and Company Quaternary structure of hemoglobin: 2 

70. Copyright (c) by W. H. Freeman and Company T-state  R-state transition in hemoglobin: structural rearrangement

71. Copyright (c) by W. H. Freeman and Company O2 binding triggers a cascade of structural rearrangements

72. Copyright (c) by W. H. Freeman and Company Concerted or Sequential cooperativity for hemoglobin? zBoth! z3 sites occupied: R-state with 4 th site having 20-fold higher affinity for O 2 z1 site occupied: T-state with other sites having 4-fold higher affinity for O 2

73. Copyright (c) by W. H. Freeman and Company A natural allosteric inhibitor of hemoglobin: 2,3-BPG

74. Copyright (c) by W. H. Freeman and Company 2,3-BPG binds to the central cavity of deoxyhemoglobin (T state) => Reduces affinity for O 2 in the T-state

75. Copyright (c) by W. H. Freeman and Company Fetal hemoglobin presents a lower affinity for 2,3-BPG  2  -chains instead of 2  -chains  mutations His  Ser in  -chains z higher affinity for O 2

76. Copyright (c) by W. H. Freeman and Company Effect of pH and pCO 2 on O 2 release from hemoglobin: Bohr effect (1904)

77. Copyright (c) by W. H. Freeman and Company Protons stabilize the quaternary structure of deoxyhemoglobin Salt bridges at acidic pH, locks T-state conformation

78. Copyright (c) by W. H. Freeman and Company Carbamylation of terminal amines by CO 2 zNegative charges at N-termini form new salt bridges zStabilize deoxyhemoglobin: favors release of O 2

79. Copyright (c) by W. H. Freeman and Company Next Lecture (No.4) zProtein synthesis (Ch. 28) zProtein analyses (Ch. 4)

80. Copyright (c) by W. H. Freeman and Company Remarks after the lecture ztoo long: 2h30 and section on hemoglobin not treated!