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Lecture 17

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. Terms to review for enzymes. Cofactor Coenzyme Prosthetic group Holoenzyme Apoenzyme Lock and Key Transition analog model Induced fit

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Lecture 17

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

  6. RNAse mechanism • 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 Page 499

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

  9. Covalent catalysis • Made up of three stages • The nucleophilic reaction between the catalyst and the substrate to form a covalent bond. • The withdrawal of electrons from the reaction center by the now electrophilic catalyst • 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. Examples structures Side chain Chymotrypsin Trypsin Elastase acetylcholinesterase NH O Serine RC-O-CH2-CH COO- (acyl ester) Phosphoglucomutase Alkaline phosphatase Serine O NH -O-P-O-CH2-CH O COO- (phosphoryl ester)

  12. Covalent Catalysis Examples structures Group Papain 3-PGAL-DH NH O Cysteine RC-S-CH2-CH COO- (acyl cysteine) CH Succinate thiokinase Histidine O COO- NH -O-P-N O (phosphoryl imidazole)

  13. Covalent Catalysis Examples structures Group Aldolase Transaldolase R' NH Lysine R-C=N-(CH2)4-CH COO- (Schiff base)

  14. Metal ion catalysis • Almost 1/3 of all enzymes use metal ions for catalytic activity. 2 main types: • Metalloenzymes-have tightly bound metal ions, mmost commonly transition metal ions such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or Co3+ • Metal-activated enzymes-loosely bind metal ions form solution-usually alkali or alkaline earth metals-Na+, K+, Ca2+

  15. Metal ion catalysis • Three ways for catalysis • Binding to substrates to orient them properly for the reaction • Mediating oxidation-reduction reactions through reversible changes in the metal ion’s oxidation state • 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. O CH3 O- C O CH3 O NO2 C p-Nitrophenylacetate H2O Chymotrypsin 2H+ -O NO2 + Acetate p-Nitrophenolate

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

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

  20. O O CH3 CH3 O-Enzyme O- C C O CH3 O NO2 C + Enzyme Chymotrypsin p-Nitrophenylacetate FAST -O NO2 p-Nitrophenolate Acyl-enzyme intermediate H2O SLOW 2H+ + Enzyme Acetate

  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 CH(CH3)2 (active Ser)-CH2OH O + • Identified catalytically important residues by chemical labeling studies. • Ser195-identified using diisopropylphospho-fluoridate (DIPF) • Irreversible! Diisopropylphospho-fluoridate (DIPF) F-P=O O CH(CH3)2 CH(CH3)2 O DIP-enzyme -P=O (active Ser)-CH2O O CH(CH3)2

  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. O Identification of catalytic residues CH2 O NH C CH2Cl S CH3 CH O Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

  25. Figure 15-19 Reaction 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 15-20a X-Ray structure of bovine trypsin.(a) A drawing of the enzyme in complex. Page 518

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

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

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

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

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

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