exam 2a 2009 - answers

exam 2a 2009 - answers - Problem 1 (17 pts) A) 2 pts Which...

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Problem 1 (17 pts) A) 2 pts Which of the following sequence would chymotrypsin hydrolize? 1. GLY-ALA-ASP-LYS-ALA 2. ARG-VAL-SER-CYS-GLN 3. ALA-GLY-PHE-ASN-MET 4. none B) A team of researcher has discovered 2 new serine proteases SERPRO1 and SERPRO2 highly homologous to chymotrypsin. The molecular basis of substrate specificity was explored using site-directed mutagenesis and kinetics studies. Mutant SERPRO is a variant of SERPRO1 where a few of the amino acids have been changed Kinetics experiments were performed for each enzyme with two different substrates (SUB1 and SUB2) Assume for all of these enzymes that K m is approximately equal to K ES (the dissociation equilibrium constant for the ES complex). 1) 2 pts Which enzyme has the largest catalytic turnover for SUB1? Why did you give that answer? SERPRO2 – highest k cat 2) 2 pts Which substrate (SUB1 or SUB2) does the mutant SERPRO1 bind the most tightly? Why did you give that answer? SUB2 – lower K M 3) 4 pts For which substrate does each enzyme has higher preference (specificity)? (give one substrate for each enzyme) SERPRO1: SUB2 SERPRO2: SUB1 Mutant SERPRO1: SUB1 Which kinetics parameter (k cat , K M or k cat /K M ) did you use to determine preference? Because higher k cat /K M 4) 4 pts Based on these observations, what are the effects of mutations in SERPRO1? Explain your answer for full credit The specificity of the enzyme was altered because k cat /K M is higher for SUB1 than SUB2 in the mutant. Mutant SERPRO1 behaves more like SERPRO2. 5) 3 pts Based on your knowledge of chymotrypsin mechanism, postulate where mutations in SERPRO1 were most likely performed? A) Catalytic triad E) Transition state B) Oxyanion hole F) P-loop C) Specificity pocket G) All of the above D) Chymotrypsogen H) None of the above SUB1 SUB2 k cat (s -1 ) K M (nM) k cat /K M (s -1 /nM) k cat (s -1 ) K M (nM) k cat /K M (s -1 /nM) Wild Type SERPRO1 0.018 0.35 0.51 77 0.023 3348 Mutant SERPRO1 43 0.19 226 0.32 0.39 0.82 Wild Type SERPRO2 50 0.08 625 0.2 0.13 1.5
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Name (print, no nicknames) _______ Key (Version A)__Spring 2009_________________________________ ‐ 2 ‐ Total_____________ Problem 2 (8 pts) Below is a double reciprocal (Lineweaver-Burk) plot, 1/V o vs. 1/[Substrate] for an enzyme in the absence of any inhibitor, and in the presence of an inhibitor. A. (4 pts) What type of inhibition is demonstrated in this plot? 1) Reversible inhibitor: Noncompetitive inhibition 2) Reversible inhibitor: Competitive inhibition 3) Reversible inhibitor: Uncompetitive inhibition 4) Irreversible inhibitor Briefly, why did you give that answer (no credit without correct reason) Same V max and different K M B. (4 pts) What is K M for this enzyme in the absence of inhbitor ? Indicate how you obtained your answer. (No credit without explanation) State units of answer . 0.2 mM = – 1
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exam 2a 2009 - answers - Problem 1 (17 pts) A) 2 pts Which...

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