Name Student number. UNIVERSITY OF GUELPH CHEM 4540 ENZYMOLOGY Winter 2002 Quiz #1: February 14, 2002, 11:30 13:00 Instructor: Prof R.

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1 UNIVERSITY OF GUELPH CHEM 4540 ENZYMOLOGY Winter 2002 Quiz #1: February 14, 2002, 11:30 13:00 Instructor: Prof R. Merrill Instructions: Time allowed = 90 minutes. Total marks = 30. This quiz represents 15% of the final grade. Please write all your answers in ink (not red ink). No examination materials may be removed from the examination room. Answers to parts A and B are to be written directly on this question booklet. Answers to part C are to be written in the answer booklet provided. Part A. Multiple-choice questions; circle the letter corresponding to the best answer. Twenty (10) questions x 1 mark per question = 10 marks total. No marks will be deducted for incorrect answers. Correct answers are indicated by the ( ) symbol. 1. Which of the following statements is false? A) A reaction may not occur at a detectable rate even though it has a favorable equilibrium B) At the end of an enzyme-catalyzed reaction, the functional enzyme becomes available to catalyze the reaction again. C) Substrate binds to an enzyme's active site. D) For S P, a catalyst shifts the reaction equilibrium to the right. E) Lowering the temperature of a reaction will lower the reaction rate. 2. Enzymes differ from other catalysts in that enzymes: A) lower the activation energy of the reaction catalyzed. B) fail to influence the equilibrium point of the reaction. C) form an activated complex with the reactants. D) usually display specificity toward a single reactant. E) are not consumed in the reaction. 3. The steady state assumption, as applied to enzyme kinetics, implies: A) K m = K s. B) the maximum velocity occurs when the enzyme is saturated. C) the ES complex is formed and broken down at equivalent rates. D) the K m is equivalent to the cellular substrate concentration. E) the enzyme is regulated. 1

2 4. Briggs and Haldane assumed that the overall scheme for an enzyme-catalyzed reaction could be written as k 1 k 2 E + S jkl ES P k 1 Using this reaction, the rate of formation of the enzyme-substrate complex can be described by the expression: A) k 1 ([E t ] [ES])[S]. B) k 1 [ES] + k 2 [ES]. C) k 2 [ES]. D) k 1 [ES]. E) k 1 ([E t ] [ES]). 5. To determine K m from the Hanes-Woolf transformation of the Michaelis-Menten equation you would: A) take the x-axis intercept where V 0 = 1/2 V max. B) take the reciprocal of the x-axis intercept. C) multiply the x-axis intercept by 1. D) take the reciprocal of the y-axis intercept. E) multiply the y-axis intercept by In the coupled enzyme system shown below, in order to measure the kinetics of E 1 the following conditions should be established: 1 A E B 2 B E C Reaction 1 Reaction 2 A) B and C must be in excess B) A must be in excess C) E 2 must be in excess D) A and B must be in excess E) none of the above 7. The term, molecular activity, refers to: A) the number of moles of substrate transformed into product per unit time per mole of enzyme active site B) the maximum velocity of a reaction over the number of moles of substrate C) the number of moles of substrate transformed into product per unit time per mole of enzyme D) the initial velocity of the catalyzed reaction at low [S]. E) none of the above. 2

3 8. An uncompetitive inhibitor is: A) a substance that binds either to the free enzyme or to the E-S complex B) a substance that binds to the E-S complex only C) a substance that binds to the free enzyme only D) a substance that causes a change in the V max only for the reaction E) none of the above 9. In the simple ph dependence model for Michaelis-Menten enzymes as described in the lecture notes, v o = V max '[S]/(K M ' + [S]) then V max ' = V max /f 2 where f 2 = A) [H + ]/K E K E2 /[H + ] B) [H + ]/K ES K ES2 /[H + ] C) [H + ]/K ES1 + K ES2 /[H + ] D) [H + ]/K E1 + K E2 /[H + ] E) none of the above. 10. The specificity constant, k cat /K M is a useful parameter for comparing the catalytic efficiencies of enzymes with substrates because: A) it is a measure of how rapidly an enzyme can work at low [S] B) it is a first order rate constant that relates [S] to reaction velocity C) it is a measure of how rapidly an enzyme can work at high [S] D) it indicates how rapidly an enzyme collides with its substrate E) none of the above Part B. Short answer/calculation questions. Answer the following (five) questions with a short answer consisting of a few sentences or less (your answer can be in point form). Each question is worth 2 marks. Five (5) questions x 2 marks each = 10 marks total. 11. Which class of enzyme of the six main classes catalyzes the following reactions? 3

4 12. Why must initial velocities be used for enzyme-catalyzed reactions when determining the kinetic parameters of an enzyme that obeys Michaelis-Menten kinetics? 13. For a single-substrate enzyme-catalyzed reaction, double reciprocal plots were determined for three different enzyme concentrations. Which of the following three families of curves (curves 1, 2 or 3) would you expect to be obtained for an enzyme that obeys Michaelis-Menten kinetics? Explain. 1) 2) 3) 4

5 14. A simple Michaelis-Menten enzyme, in the absence of any inhibitor, displayed the following kinetic behaviour. The expected value of V max is shown on the y-axis. (a) Sketch a double-reciprocal plot that corresponds to the velocity-versus-substrate curve. (b) Provide an explanation for the kinetic results. 15. In the conversion of A into D in the following biochemical pathway, enzymes E A, E B, and E C have the K M values indicated under each enzyme. If all of the substrates and products are present at a concentration of 10-4 M, which step will be rate-limiting and why? E A E B E C A B C D K M = 10-2 M 10-4 M 10-5 M 5

6 Part C. Problems-based questio ns. Answer the following three (3) questions in the examination bookl et provided. Please write your answers in pen (not red ink). Each question is worth 3, 3, and 4 marks, respectively. Three ( 3) questions (3, 3, and 4 marks) = 10 marks total. 16. The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic reactions such as the synthesis of DNA. This hydrolytic reaction is catalyzed in E. coli by a pyrophosphatase that has a mass of 120 kda and consists of six identical subunits. For this enzyme, a unit of activity is defined as the amount of enzyme that hydrolyzes 10 µmol of pyrophosphate in 15 minutes at 37 C under standard assay conditions. The purified enzyme has a V max of 2800 units per milligram of enzyme. (a) How many moles of substrate are hydrolyzed per second per milligram of enzyme when the substrate concentration is much greater than K M? (b) How many moles of active site are there in 1 mg of enzyme? (c) What is the turnover number of the enzyme? 17. Human carbonic anhydrase was tested at a bicarbonate concentration of 3 x 10-7 M, the K M for the substrate is 2 x 10-5 M. After 4 minutes, 13% of the substrate was converted to product. (a) What percent of the substrate will be CO 2 product after 8 minutes? (b) What is the V max given these conditions? 18. Malonate, a competitive inhibitor of succinate dehydrogenase, was found to cause 95% inhibition of the enzyme s activity. (a) If the succinate (substrate) concentration for the enzyme was 3.5 x 10-5 M and the K M for this succinate is 4.4 x 10-6 M, what was the initial malonate concentration (K i = 2.4 x 10-7 M)? (b) What concentration must succinate be raised to in order to restore the velocity of the enzyme to its uninhibited value? Have a nice Break Week! 6