1. Lecture 17 –Exams in Chemistry office with M’Lis. Please show your ID to her to pick up your exam. –Quiz on Friday –Enzyme mechanisms

2. General Acid-Base Catalysis General acid catalysis- a process in which partial proton transfer from a Brønstead acid (a species that can donate protons) lowers the free energy of a reaction’s transition state. General base catalysis - process in which partial proton abstraction by a Brønstead base (a species that can combine with a proton) lowers the free energy of a reaction’s transition state. General acid-base catalysis-a combination of both.

3. Figure 15-1aMechanisms of keto–enol tautomerization. (a) Uncatalyzed. Page 497

4. Figure 15-1bMechanisms of keto–enol tautomerization. (b) General acid catalyzed. Page 497

5. Figure 15-1cMechanisms of keto–enol tautomerization. (c) General base catalyzed. Page 497

6. General Acid Base Catalysis Ex. Ester hydrolysis + H +  OR C O H H2OH2O C O H O H H + +  C O  C O H O H H+H+ OH C O + ROH - H +

7. General Acid-Base Catalysis Large number of possible amino acids Requires that they can accept and donate a proton Glu, Asp Lys, His, Arg Cys, Ser, Thr Also can include metal cofactors Example can be observed in carboxypeptidase A (both acid and base catalysis)

8. General Acid-Base Catalysis Ex. Carboxypeptidase A Zn ++ O    R C N H H-C CO 2 - O H-C-R NH C O + Arg 145 HO-Tyr 248 Glu 72 + Arg  H H C-O - O Glu 270 His 69 His 196 Key aas that holds molecule in place Tyr also plays role as 2 nd acid catalyst Zn plays role of acid (4 th ligand is normally H 2 O, but it is displaced by peptide binding) Glu acts as base catalyst to polarize water and form nucleophile

9. Study of Enzyme Mechanisms X-ray crystallography-crystallize the molecule with substrate in place and compare to crystal structure of the molecule without the substrate (differences in structure) For carboxypeptidase A they could show that Water is expelled by binding of substrate Arg145 moves 2Å closer to the carboxyl group of the substrate Glu270 moves 2Å towards the C=O group Tyr248 moves 12Å towards the amide plane of the peptide Also able to show what aa surround certain groups- Tyr248 in a hydrophobic pocket.

10. Study of Enzyme Mechanisms Check the pH profile of the enzyme. For carboxypeptidase The coordination of Zn by His69 and His196 (pK 6.0) Tyr248 (pK 9.1) 9876 6.7 8.5 pH Log (V max /K M ) Example in book: RNAse (p. 499)

11. Lysozyme (Strain and Acid Catalysis) Hen Egg White (HEW) Lysozyme digests bacterial cell walls. Cleaves  (1 4) glycosidic linkages from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) Requires about 6 sugars for good recognition. SugA-SugB-SugC-SugD-SugE-SugF

12. Lysozyme (Strain and Acid Catalysis) In theory SugA-SugB-SugC-SugD-SugE-SugF H+H+ O OH O O D E : O C OR  Must distort ring into a flat, planar shape Supply acid catalysis Asp 52 C O -O-O Glu 35 C OH O

13. Lysozyme (Strain and Acid Catalysis) In practice

14. Figure 15-12Interactions of lysozyme with its substrate. Page 510

15. Figure 15-11Chair and half- chair conformations. Page 510

16. Study of Enzyme Mechanisms Lysozyme Only the D ring is strained Glu35 is in a hydrophobic environment Asp52 is in a hydrophilic environment Covalent modification of the active site Block essential groups May or may not act at active site Cd or R-As=O (trivalent arsenic)

17. The Aspartate Proteases Pepsin, Renin, HIV protease (AZT targets this) General acid-base catalysis

18. Serine hydrolases: trypsin, chymotrypsin, elastase Synthesized in pancreas as inactive zymogen (ex. trypsinogen) Generally operate by "charge relay system" Asp102, His57, Ser195 conserved in all 3 enzymes. Asp 102 NH COO N His 57 H Ser 195 O H R NHNH C R' O - 1

19. Serine hydrolases: trypsin, chymotrypsin, elastase Asp 102 NH COO N His 57 Ser 195 O : R NHNH C R' O H - 2 Rate limiting step for amides Asp 102 NH COO N His 57 H - Ser 195 O RNH 2 C R' O

20. Serine hydrolases: trypsin, chymotrypsin, elastase Asp 102 NH COO N His 57 H - Ser 195 O C R' O O H H Asp 102 NH COO N His 57 Ser 195 O H O C R' O H - 3 Rate limiting step for esters

21. Serine hydrolases: trypsin, chymotrypsin, elastase Asp 102 NH COO N His 57 H - Ser 195 O H H O C R' O

22. Charge-relay systems Relay charges between amino acid side chains in order to catalyze the reaction.

23. Summary: various methods to increase rate Increase frequency of the correct group in the correct place e.g. proximity effect Lower E A by specific catalysis -acid-base catalysis, nucleophile or electrophile Raise energy of reactants (closer to E A ) - ring distortion, transition state analog Provide alternate low E A pathway - covalent catalysis.] Michaelis Menten Lineweavear Burk Eadie Hofstee Competitive inhibition Noncompetitive inhibition

24. Terms to review for enzymes Cofactor Coenzyme Prosthetic group Holoenzyme Apoenzyme Lock and Key Transition analog model Induced fit Active site, binding site, recognition site, catalytic site

25. Catalytic Mechanisms Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation effects (ex. anhydride) Preferential binding of the transition state complex

26. General Acid-Base Catalysis Large number of possible amino acids Requires that they can accept and donate a proton Glu, Asp Lys, His, Arg Cys, Ser, Thr Also can include metal cofactors (metal ion catalysis) Example can be observed in RNAse

27. Figure 15-2The pH dependence of V¢ max /K¢ M in the RNase A–catalyzed hydrolysis of cytidine-2¢,3¢ -cyclic phosphate. Page 499 Example in book: RNAse (p. 499)

28. Page 499 His12 acts as general base-takes proton from RNA 2’-OH-making a nucleophile which attacks the phosphate group. His119 acts as a general acid to promote bond scission. 2’,3’ cyclic intermediate is hydrolyzed through the reverse of the first step-water replaces the leaving group. His12 is the acid, His119 acts as the base RNAse mechanism

29. Covalent catalysis Rate acceleration through the transient formation of a catalyst-substrate covalent bond. Example-decarboxylation of acetoacetate by primary amines Amine nucleophilically attacks carbonyl group of acetoacetate to form a Schiff base (imine bound)

30. Figure 15-4The decarboxylation of acetoacetate. Page 500 e - sink uncatalyzed Catalyzed by primary amine

31. Covalent catalysis Made up of three stages 1.The nucleophilic reaction between the catalyst and the substrate to form a covalent bond. 2.The withdrawal of electrons from the reaction center by the now electrophilic catalyst 3.The elimination of the catalyst (reverse of 1.) Nucleophilic catalysis - covalent bond formation is limiting. Electrophilic catalysis-withdrawal of electrons is rate limiting

32. Covalent catalysis Nucleophilicity is related to basicity. Instead of abstracting a proton, nucleophilically attacks to make covalent bond. Good covalent catalysts must have high nucleophilicity and ability to form a good leaving group. Polarized groups (highly mobile e-) are good covalent catalysts: imidazole, thiols. Lys, His, Cys, Asp, Ser Coenzymes: thiamine pyrophosphate, pyridoxal phosphate.

33. Covalent Catalysis Form transient, metastable intermediates that can supply bond energy into the reaction. Serine Side chain NH RC-O-CH 2 -CH O COO- (acyl ester) Chymotrypsin Trypsin Elastase acetylcholinesterase structures Examples Serine - O-P-O-CH 2 -CH O (phosphoryl ester) O NH COO- Phosphoglucomutase Alkaline phosphatase

34. Covalent Catalysis Cysteine Group NH RC-S-CH 2 -CH O COO- (acyl cysteine) Papain 3-PGAL-DH structures Examples Histidine - O-P-N O (phosphoryl imidazole) O NH COO- Succinate thiokinase CH

35. Covalent Catalysis Lysine Group NH R-C=N-(CH 2 ) 4 -CH R' COO- (Schiff base) Aldolase Transaldolase structures Examples

36. Metal ion catalysis Almost 1/3 of all enzymes use metal ions for catalytic activity. 2 main types: 1.Metalloenzymes-have tightly bound metal ions, mmost commonly transition metal ions such as Fe 2+, Fe 3+, Cu 2+, Zn 2+, Mn 2+, or Co 3+ 2.Metal-activated enzymes-loosely bind metal ions form solution-usually alkali or alkaline earth metals-Na +, K +, Ca 2+

37. Metal ion catalysis Three ways for catalysis 1.Binding to substrates to orient them properly for the reaction 2.Mediating oxidation-reduction reactions through reversible changes in the metal ion’s oxidation state 3.Electrostatically stabilizing or shielding negative charges.

38. Serine Hydrolases (Proteases) Chymotrypsin, trypsin and elastase. All have a reactive Ser necessary for activity. Catalyze the hydrolysis of peptide (amide) bonds. Chymotrypsin can act as an esterase as well as a protease. Study of esterase activity provided insights into the catalytic mechanism.

39. NO 2 p-Nitrophenylacetate CH 3 O O C O-O- O C NO 2 -O-O p-Nitrophenolate Acetate Chymotrypsin H2OH2O 2H + +

40. Serine Hydrolases (Proteases) Reaction takes place in 2 phases 1.The “burst phase”-fast generation of p- nitrophenolate in stoichiometric amounts with enzyme added 2.The “steady-state phase”-p-nitrophenolate generated at reduced but constant rate; independent of substrate concentration.

41. Figure 15-18Time course of p- nitrophenylacetate hydrolysis as catalyzed by two different concentrations of chymotrypsin. Page 516

42. NO 2 p-Nitrophenylacetate CH 3 O O C O-Enzyme O C NO 2 -O-O p-Nitrophenolate Acyl-enzyme intermediate Chymotrypsin H2OH2O 2H + + Enzyme CH 3 O-O- O C Acetate + Enzyme SLOW FAST

43. Chymotrypsin Follows a ping pong bi bi mechanism. Rate limiting step for ester hydrolysis is the deacylation step. Rate limiting step for amide hydrolysis is first step (enzyme acylation).

44. Identification of catalytic residues Identified catalytically important residues by chemical labeling studies. Ser195-identified using diisopropylphospho- fluoridate (DIPF) Irreversible! (active Ser)-CH 2 OH F-P=O O Diisopropylphospho -fluoridate (DIPF) O + CH(CH 3 ) 2 (active Ser)-CH 2 O -P=O O O CH(CH 3 ) 2 DIP-enzyme

45. Identification of catalytic residues His57 was identified through affinity labeling Substrate analog with a reactive group that specifically binds to the active site of the enzyme forms a stable covalent bond with a nearby susceptible group. Reactive substrate analogs are sometimes called “Trojan horses” of biochemistry. Affinity labeled groups can be identified by peptide mapping. For chymotrypsin, they used an analog to Phe.

46. CH 2 Cl CH 3 C O NH S O O CH CH 2 Identification of catalytic residues Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

47. Figure 15-19Reaction of TPCK with chymotrypsin to alkylate His 57. Page 517

48. Homology among enzymes Bovine chymotrypsin, bovine trypsin and porcine elastase are highly homologous ~40% identical over ~240 residues. All enzymes have active Ser and catalytically essential His X-ray structures closely related. Asp102 buried in a solvent inaccessible pocket (third enzyme in the “catalytic triad”)

49. X-ray structures explain differences in substrate specificity Chymotrypsin - bulky aromatic side chains (Phe, Trp, Tyr) are preferred and fit into a hydrophobic binding pocket located near catalytic residues. Trypsin - Residue corresponding to chymotrypsin Ser189 is Asp (anionic). The cationic side chains of Arg and Lys can form ion pairs with this residue. Elastase - Hydrolyzes Ala, Gly and Val rich sequences. The specificity pocket is largely blocked by side chains of Val and a Thr residue that replace Gly residues that line the binding pocket of chymotrypsin and trypsin.

50. Figure 15-20aX-Ray structure of bovine trypsin. (a) A drawing of the enzyme in complex. Page 518

51. Figure 15-20bX-Ray structure of bovine trypsin. (b) A ribbon diagram of trypsin. Page 519

52. Figure 15-20cX-Ray structure of bovine trypsin. (c) A drawing showing the surface of trypsin (blue) superimposed on its polypeptide backbone (purple). Page 519

53. Figure 15-21The active site residues of chymotrypsin. Page 520

54. Figure 15-22Relative positions of the active site residues in subtilisin, chymotrypsin, serine carboxypeptidase II, and ClpP protease. Page 521

55. Figure 15-23 Catalytic mechanism of the serine proteases. Page 522