The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 2 Group Transfer Reactions: Hydrolysis, Amination, Phosphory

1. The Organic Chemistry of Enzyme-Catalyzed ReactionsChapter 2Group Transfer Reactions: Hydrolysis, Amination, Phosphorylation

2. Hydrolysis ReactionsAmide Hydrolysis Peptidases (proteases if protein hydrolysis involved) catalyze the hydrolysis of peptide bonds

3. Reaction catalyzed by peptidases scissile bond Scheme 2.1

4. Classifications of peptidases Figure 2.1

5. Endopeptidases • Representative example is -chymotrypsin • Regiospecifically hydrolyzes peptide bonds of the aromatic acids • P1 -chymotrypsin is Phe, Tyr, and Trp • P1 for trypsin is Arg and Lys

6. Endopeptidase Mechanism for -chymotrypsin showing catalytic triad Scheme 2.2

7. Evidence for Acyl Intermediate Reaction of chymotrypsin with p-nitrophenyl acetate: demonstration of an initial burst Use of an alternate, poor substrate to change the rate-determining step Figure 2.2

8. Typical enzyme reaction in which the first step is fast Scheme 2.3

9. Evidence for formation of an acyl intermediate Reaction of -chymotrypsin with aryl cinnamate esters common acyl intermediate Scheme 2.4 Enzymatic rates - same Nonenzymatic rates - different

10. Formation of an acyl intermediate in the reaction catalyzed by -chymotrypsin To demonstrate covalent intermediate: pH 5 pH 8 pH optimum stops here below pH optimum for catalysis kinetically competent Scheme 2.5

11. Gel Filtration (aromatic amino acids in enzyme) excess substrate Figure 2.3

12. Reactivation of acetylchymotrypsin by hydroxylamine To support formation of acetylchymotrypsin reactivated enzyme Isolate and characterize Scheme 2.6

13. Rate of base hydrolysis of acetylchymotrypsin denatured by 8 M urea is identical to rate of base hydrolysis in 8 M urea with a model compound, O-acetylserinamide

14. Reaction of -chymotrypsin with an organophosphofluoridate affinity labeling agent To show involvement of a serine residue at the active site affinity labeling agent Scheme 2.7

15. Kinetics of affinity labeling of enzymes Affinity labeling agent substrate protection Scheme 2.8

16. Irreversible inhibitors exhibit time-dependent inhibition Reaction after E•I complex formation is rate limiting; therefore, time dependent

17. Enzyme Inactivation Correlation between loss of enzyme activity and incorporation of radioactivity during enzyme inactivation loss of enzyme activity and incorporation of radioactivity correspond (1 : 1 inactivator : enzyme) Figure 2.4 With [32P] get 1 equiv 32P bound to enzyme; 6 N HCl at 110 °C, 24 h gives [32P]phosphoserine Peptidase hydrolysis gives [32P]peptide containing modified Ser-195.

18. Evidence for Histidine Participation inactivator (TPCK) substrate With [14C]TPCK get 1 equiv. [14C] bound; pepsin hydrolysis gives a [14C] peptide with His-57 modified

19. Mechanism of inactivation of -chymotrypsin by -chloromethyl ketones -chymotrypsin (side reaction) (S)-N-Ac-L-Ala-L-Phe No hydrolysis product in absence of enzyme (nonenzyme control) Same stereochemistry as 2.13 Evidence against a single SN2 reaction

20. Double inversion mechanism for inactivation of serine proteases by -chloromethyl ketones Scheme 2.10

21. Three possible mechanisms for inactivation of -chymotrypsin by -chloromethyl ketones inversion of configuration Scheme 2.11 overall retention of configuration

22. -Chymotrypsin was inactivated by 2.20, and X-ray crystal structure showed His-57 alkylated with stereochemistry retained

23. Evidence for Deacylation Mechanism acetyl-serine model General base catalysis by imidazole solvent 2H isotope effect 2-3

24. Model study for deacylation step Ser mimic His mimic not active kH2O/kD2O = 3 Addition of PhCOO- as a model of Asp-102 increases rate 2500 fold

25. Chemical model for the deacylation step in -chymotrypsin 1/18 rate of chymotrypsin Improved model general base catalysis Scheme 2.12

27. Aspartate Protease Proposed mechanism for HIV-1 protease Note: General acid-base catalysis, not covalent catalysis Scheme 2.14

28. Carboxypeptidases (an exopeptidase) Affinity labeling agent for CPA labels Glu-270

29. General base catalytic mechanism for carboxypeptidase A Scheme 2.15 Zn++ is a cofactor

30. Nucleophilic mechanism for carboxypeptidase A Scheme 2.16 Not detected or trapped

31. Principle of Microscopic Reversibility For any reversible reaction, the mechanism in the reverse direction must be identical to that in the forward reaction (only reversed) This can be a valuable approach to study enzyme mechanisms.

32. Reverse of the general base mechanism Reverse of general base catalytic reaction of carboxypeptidase A in the presence of H218O Scheme 2.17 Requires amino acid to release H218O

33. Reverse of nucleophilic catalytic reaction of carboxypeptidase A in the presence of H218O Reverse of the nucleophilic mechanism Does not require amino acid to release H218O Scheme 2.18 Found amino acid is required for H218O release (general base mechanism)

34. Alternative mechanism for carboxypeptidase A on the basis of the X-ray structure with a ketone bound From Crystal Structure of Ketone Scheme 2.19 • Functions of Zn++ Cofactor • Coordinate to H2O to make it more nucleophilic • Coordinate to carbonyl to make it more electrophilic

35. Typical esterase mechanism Scheme 2.20 Covalent catalytic mechanism

36. Mechanism for acetylcholinesterase no anion cluster of aromatic residues instead Scheme 2.21 (cation- complex) Catalytic triad has a Glu instead of an Asp

37. Favored enantiomer substrate for lipases

38. An example of the enantioselectivity of lipases/esterases Scheme 2.22 Useful for chiral resolutions of alcohols

39. Catalytic Antibodies (abzymes) • Antibodies are proteins that scavenge macromolecular xenobiotics • Form very tight complexes with macromolecule, which causes • a cascade of events, leading to degradation of macromolecule • A catalytic antibody is an antibody that catalyzes a chemical reaction

40. Construction of Catalytic Antibodies • A transition state analogue that mimics the transition state of • the desired reaction is synthesized--called a hapten • Hapten is attached to a carrier molecule capable of eliciting • an antibody response--called an antigen • Antigen injected into a mouse or rabbit • Monoclonal antibodies (ones that bind to one region of the • antigen) are isolated for that antigen • The monoclonals are tested for catalytic activity

41. Transition State Analogue Inhibitor • Inhibitor molecules resembling the transition-state species should bind to enzyme much more tightly than the substrate • Therefore, a potent enzyme inhibitor would be a stable compound whose structure resembles that of the substrate at a postulated transition state--a transition state inhibitor

42. Comparison of an ester hydrolysis tetrahedral intermediate and a phosphonate “transition state” mimic Development of Catalytic Antibodies Figure 2.5

43. mimics tetrahedral intermediate in ester hydrolysis X = OH hapten X = macromolecule antigen (elicits antibody response)

44. Two different monoclonal antibodies raised, each catalyzes hydrolysis of different epimer R1 = Bn R2 = H R1 = H R2 = Bn

45. Aminations

46. Glutaminase activity (generation of NH3) A covalent catalytic mechanism for the “glutaminase” activity of glutamine-dependent enzymes Scheme 2.23 • Free NH3 is toxic to cell - this protects cell from NH3 • NH3 can be substituted for Gln, but Km 102-103  higher

47. Evidence for -glutamyl enzyme intermediate in glutamine-dependent enzyme Evidence for covalent catalysis Scheme 2.24

48. Comparison of the structure of the -chloromethyl ketone of asparagine with the structure of glutamine irreversible inhibitor substrate Figure 2.6

49. modify Cys residue Blocks enzyme reaction with Gln, but not with NH3; therefore 2 binding sites

50. Mechanism-based inactivators of Gln-dependent enzymes • Mechanism-based inactivator • Unreactive compound whose structure resembles • the substrate (or product) for an enzyme • Acts like a substrate and is converted into a species • that inactivates the enzyme • Cannot escape enzyme until it inactivates it