MCB354SP09_ExamIIKEY (1) - Name Answer the following...

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Name: ______________________ 1 Answer the following questions in the space provided. Please write legibly! Write your name at the top of each page. Dr. Spies’ material Constants: R=1.96 cal.K -1 .mol -1 ; k (Boltzmann constant) = 1.99 cal.K -1 .mol -1 1. (25 points) The kinetics of an enzyme are measured as a function of the substrate concentration in the presence and absence of 2 X 10 -3 M of an inhibitor. The V max and the K M , in the absence of inhibitor were determined to be 47.6 _M/min and 1.1 X 10 -5 M, respectively. Velocity measurements with increasing concentrations of inhibitor showed no change in the V max . [s] M Velocity, no inhibitor (_M/min) Velocity with inhibitor (_M/min) 0.3 X 10 -5 10.4 4.1 0.5 X 10 -5 14.5 6.4 1.0 X 10 -5 22.5 11.3 3.0 X 10 -5 33.8 22.6 9.0 X 10 -5 40.8 33.8 A) Calculate K M (apparent) at 2 X 10 -3 M Inhibitor Ans: Use MM equation; have V max , so choose a substrate concentration and initial velocity from the table and calculate K m (apparent) e.g., vo/V max = [s]/([s] + K m (app)); for the [s] = 1 X 10 -5 M vo= 11.3 _M/min K m (app) = 3.2 X 10 -5 M B) What type of inhibition is this? Ans: Competitive C) What is the binding constant of the inhibitor ( i.e. , equilibrium dissociation constant)? Ans: Use K m (app) = K m (1 + [I]/[K i ]); only unknown is K i K i = [I]/((K m (app)/K m ) - 1) = 1.1 X 10 -3 M D) If [s] = 1X10 -5 M, and [I] = 2 X 10 -3 M, what fraction of enzyme molecules have substrate bound? Ans: need to determine vo/V max , so vo/V max = [s]/([s] + K m (1 + [I]/K i )) = 0.243
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Name: ______________________ 2 2. (35 points) In class we discussed the use of para-nitrophenolacetate as an alternative substrate for chymotrypsin. The overall reaction is shown below: A) Write out the detailed mechanism for enzymatic hydrolysis of this substrate. Show all key active site residues. Make sure to use correct mechanistic notation. Ans : Show catalytic triad (Asp, His, Ser); deprotonation of Ser; attack of Ser oxyanion on carbonyl of pNP-acetate; tetrahedral intermediate (stabilization by oxyanion hole); protonation of leaving hydroxyl by His; show esterified intermediate; activation of water by His abstracting proton, attack of ester carbonyl carbon by hydroxide; new tetrahedral intermediate (oxyanion hole stabilization); donation of proton to serine oxygen; and breakdown of tetrahedral intermediate, breaking of serine oxygen to carbon to form acetic acid product. B) Indicate how chymotrypsin utilizes acid/base catalysis and covalent catalysis. Ans : the His acts as both a base and acid in both phases of the reaction. The deprotonated Ser becomes a strong nucleophile and makes a covalent bond with the substrate (covalent catalysis). C) The PNP-acetate reacts with a much slower k cat than longer, natural substrates. How might you explain this?
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