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. 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

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

4. 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

5. 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)

6. 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

7. 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)

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

9. 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

10. 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.

11. 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

12. 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

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

14. 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+

15. 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.

16. 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.

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

18. 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.

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

20. 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

21. 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).

22. 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

23. 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.

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

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

26. 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”)

27. 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.

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

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

30. 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

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

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

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