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Review Session: Monday, Dec. 8 6 pm BI 212 Exam Wednesday, Dec. 10 10:30 am FH 4

Review Session: Monday, Dec. 8 6 pm BI 212 Exam Wednesday, Dec. 10 10:30 am FH 4. Holiday Concerts: Whatcom Chorale Sunday, Dec. 14 3pm and 7:30 pm WWU PAC Concert Hall Music by Charpentier, Saint-Saens, Berlioz and some French carols.

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Review Session: Monday, Dec. 8 6 pm BI 212 Exam Wednesday, Dec. 10 10:30 am FH 4

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  1. Review Session: Monday, Dec. 8 6 pm BI 212 Exam Wednesday, Dec. 10 10:30 am FH 4

  2. Holiday Concerts: Whatcom Chorale Sunday, Dec. 14 3pm and 7:30 pm WWU PAC Concert Hall Music by Charpentier, Saint-Saens, Berlioz and some French carols.

  3. Figure 14-15 Effect of pH on the initial rate of the reaction catalyzed by the enzyme fumarase. Page 487

  4. Catalytic Mechanism Determination 1) kinetic analysis - what is the kinetic “signature”? - mode of inhibition revealed - determine rates for individual steps - does order of addition matter? (sequential vs. Ping-Pong) 2) active site modification (irreversible inhibitors) - derivatize protein; identify the modified sidechain(s) 3) structure determination (e.g. RNase A, lysozyme, serine proteases)

  5. Figure 15-3 The bovine pancreatic RNase A–catalyzed hydrolysis of RNA is a two-step process with the intermediate formation of a 2¢,3¢ -cyclic nucleotide. Reverse rxn with water as leaving group His 12: general base Nucleophillic attack His 9 protonates leaving group Page 499 = general acid/base catalysis

  6. Figure 15-2 The pH dependence of V¢max/K¢M in the RNase A–catalyzed hydrolysis of cytidine-2¢,3¢ -cyclic phosphate. Page 499

  7. Enzymatic catalysis proceeds by one or more of: 1) general acid/base catalysis (GABC) 2) covalent catalysis 3) Metal ion catalysis 4) electrostatic stabilization 5) proximity effects 6) preferential stabilization of the

  8. Covalent catalysis Is characterized by the formation of a covalent Enz-S adduct that alters the reaction pathway Nucleophiles: many amino acid side chains (H, K, C, S, D, E, Y), some coenzymes (TPP) Electrophiles: some coenzymes (e.g. PLP)

  9. Stryer Fig. 9.1

  10. Stryer Fig. 9.2 Identifying an active ser--Out of 28 ser, only #195 is labeled

  11. Figure 15-19 Reaction of TPCK with chymotrypsin to alkylate His 57. Page 517 An affinity label

  12. This assay allows the use of ester hydrolysis to help formulate a kinetic model for catalysis by chymotrypsin. Stryer Fig. 9.3 Chromogenic substrate (like V2 p. 516)

  13. Slow hydrolysis of ES Fast release of PNP Stryer Fig. 9.3 Kinetics indicate a 2-step reaction

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

  15. Figure 15-20a X-Ray structure of bovine trypsin.(a) A drawing of the enzyme in complex with substrate analog. Page 518 Similar backbone structrures for chymotrypsin and elastase.

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

  17. Figure 15-21 The active site residues of chymotrypsin. Page 520 Catalytic Triad

  18. Stryer Fig. 9.5 Covalent catalysis

  19. Figure 15-25a Transition state stabilization in the serine proteases. (a) The Michaelis complex. Page 524

  20. Figure 15-25b Transition state stabilization in the serine proteases. (b) The tetrahedral intermediate. Page 524 Preferential binding to transition state: 3 new H bonds from after distortion

  21. His 57: GB Stablilized by H bond to asp 102 Figure 15-23 Catalytic mechanism ofthe serine proteases. Page 522 Reverse: His 57: GA

  22. Table 15-4 A Selection of Serine Proteases. Page 516

  23. Specificity of ser proteases determined by different binding pockets: Chy--slit lined by hydrophobic residues Tryp- asp at bottom of pocket Elastase: Pocket blocked by val-thr midway down the pocket.

  24. 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

  25. Example of “convergent” evolution.

  26. From E. coli Figure 15-22 Relative positions of the active site residues in subtilisin, chymotrypsin, serine carboxypeptidase II, and ClpP protease. From wheat germ Page 521

  27. Figure 15-27 Activation of trypsinogen to form trypsin. Page 527

  28. Figure 15-28 Activation of chymotrypsinogen by proteolytic cleavage. Page 528

  29. Figure 15-24a Trypsin–BPTI complex. (a) The X-ray structure shown as a cutaway surface drawing indicating how trypsin (red) binds BPTI (green). Page 523

  30. Figure 15-24b Trypsin–BPTI complex. (b) Trypsin Ser 195, the active Ser, is in closer-than-van der Waals contact with the carbonyl carbon of BPTI’s scissile peptide. Page 523

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