STUDY GUIDE #2 Winter 2000 Chem 4540 ANSWERS

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1 STUDY GUIDE #2 Winter 2000 Chem 4540 ANSWERS R. Merrill 1. Sketch the appropriate plots on the following axes. Assume that simple Michaelis- Menten kinetics apply. 2. The enzyme-catalyzed hydrolysis of sucrose: sucrose + H 2 O glucose + fructose takes the following time course. Time (min) [sucrose] M Determine the first-order rate constant and the half-life of the reaction. Why does this bimolecular reaction follow a first-order rate law? How long will it take to hydrolyze 99% of the sucrose initially present? How long will it take if the amount of sucrose initially present is twice that given in the table (above)? 2. (Answer) 1

2 Time (min) [sucrose] M ln[sucrose] ln [sucrose] Slope = -3.5 x 10-3 k = -slope = min -1 t 1/2 = 0.693/k = 0.693/ min -1 = 198 min Time (min) The reaction follows pseudo first-order kinetics because in aqueous solution the H 2 O concentration of 55.5 M remains essentially constant in comparison to the sucrose concentration. The reaction actually is second order. (1-0.99) = 0.01 = (1/2) n where n is the number of half-lives n (log 0.5) = log 0.01 n = -2.00/ = 6.64 half-lives. Thus, the time to hydrolyze 99% of the sucrose = t 0.01 = nt 1/2 = 6.64 x 198 = 1315 min. The time to react a given fraction of a substance that reacts in a first-order process is independent of the amount initially present. Hence, the time to hydrolyze 99% of 2x M sucrose is also 1315 min. 3. (a) [S] 0 = 0.5 µmole/25 ml = 2 x 10-2 µmole/ml = 2 x 10-5 M K M = 6 x 10-4 M therefore [S] 0 < K M and thus first order kinetics. (b) [S] 0 = 0.5 mmole/25 ml = 2 x 10-2 mmole/ml = 2 x 10-2 M 2

3 K M = 6 x 10-4 M therefore [S] 0 > K M and thus zero order kinetics. 4. (a) We must assume that the initial velocity observed at the [S] = 0.01M is the V max, i.e., [S] 0 >> K M, therefore v 0 = V max = 35 µmole L -1 min -1. At [S] 0 = 3.5 x 10-5 M then v 0 = (35 µmole L -1 min -1 ) (3.5 x 10-5 M)/[(2.5 x 10-5 M) + (3.5 x 10-5 M)] = 22.3µmole L -1 min -1. (b) At 4.0 x 10-4 M [S] 0, [S] 0 is between K M and 100 K M (note: at [S] 0 100K M, v 0 = V max ) Thus, v 0 will be between 17.5 µmole L -1 min -1 (V max /2) and 35 µmole L -1 min -1 (V max ) v 0 = (35 µmole L -1 min -1 ) (4 x 10-4 M)/[(2.5 x 10-5 M) + (4 x 10-4 M)] = 33.3µmole L -1 min -1. (c) At 2 x 10-4 M [S] 0 then [S] 0 = (10) K M v 0 = V max [S]/(K M + [S]) = V max (10 K M )/(K M + 10 K M ) = V max (10/11) = 0.91V max v 0 = 0.91(35 µmole L -1 min -1 ) = 31.9 µmole L -1 min -1 (d) At 2 x 10-6 M [S] 0 then [S] 0 = 0.1 K M v 0 = V max (0.1K M )/(K M K M ) = V max (0.1 K M )/1.1K M = V max v 0 = 0.091(35 µmole L -1 min -1 ) = 3.19 µmole L -1 min -1 (e) At 1.2 x 10-6 M [S] 0 : v 0 = (35 µmole L -1 min -1 ) (1.2 x 10-6 M)/[(2 x 10-5 M) + (1.2 x 10-6 M)] = 1.98 µmole L -1 min v 0 /V max = [S]/(K M + [S]) 0.90/1 = [S] 90 /(K M + [S] 90 ) 0.90 K M [S] 90 = [S] 90 ; 0.90K M = [S] [S] 90 ; 0.90K M = 0.10 [S] 90 ; [S] 90 = 0.90K M /0.10; [S] 90 = 9K M Thus, when the substrate concentration is 9K M, the v 0 is 90% of the V max. v 0 /V max = [S]/(K M + [S]) 0.10/1 = [S] 10 /(K M + [S] 10 ) 0.10K M [S] 10 = [S] 10 ; 0.10K M = [S] [S] 10 ; 0.10K M = 0.90 [S] 10 ; [S] 10 = 0.10K M /0.90; [S] 10 = 0.111K M Thus, when the substrate concentration is 0.111K M then v 0 = 10% of V max. [S] 90 /[S] 10 = 9K M /0.111K M = (a) [S] 0 = 2 x 10-5 M << K M therefore reaction is first order w.r.t. substrate concn. For a first order reaction: t 1/2 = 0.693/k, so 6 min = 0.693/k, k = 1.16 x 10-1 min -1. (b) Recall from lecture notes: k = V max /K M for a first order reaction so then V max = k(k M ) V max = (1.16 min -1 )(5 x 10-3 M) = 5.8 x 10-4 M min -1 3

4 (c) Recall Equation M-14 from notes addendum: log([s] 0 /[S] t ) = kt for a first order reaction log (2 x 10-5 M/[S] t ) = M min -1 (15 min) (log 2 x log [S] t ) = M log 2 x log [S] t = M ( ) -log [S] t = M -log [S] t = M 1/[S] t = x 10 5 [S] t = 3.54 x 10-6 M Thus, [P] 15 = [S] 0 - [S] t = (2 x 10-5 M) - (3.54 x 10-6 M) = x 10-5 M 7. (a) See graph 7(a) Lineweaver-Burk plot, y = x x 10-7 X Y intercept = 1/V max = 1.25 x 10-2 V max = 80 µmoles L -1 min -1 X intercept = -1/K M = x 10-2 /4.986 x 10-7 = x 10 4 K M = -(1/-2.51 x 10 4 ) = 4.0 x 10-5 M (b) See graph 7(b) Hanes Plot, y = x x 10-2 X Slope = 1.25 x 10-2 = 1/V max = 1/1.25 x 10-2 = 80 µmoles L -1 min -1 Y intercept = K M /V max = x 10-7, K M = 80(4.986 x 10-7 ) = 3.99 x 10-5 M (c) See graph 7(c) Eadie-Hofstee Plot, y = x 10-5 X Slope = -K M = -( x 10-5 ) = 3.99 x 10-5 M Y intercept = V max = 80 µmoles L -1 min -1 (d) See graph 7(d), y = 2.01 x x 10 4 X Eadie-Scatchard Plot Slope = -1/K M = -1/2.50 x 10 4 = 4.0 x 10-5 M = K M Y intercept = V max /K M = 2.01x 10 6, V max = 2.01 x 10 6 (4.0 x 10-5 ) = 80 µmoles/l - 1 min -1 (e) See graph 7(e), V max = µmoles L -1 min -1 ; K M = x 10-5 M. 4

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9 8. (a) [S] 0 = 1 x 10-5 M << K M (2 x 10-3 M) therefore first order and the reaction will be directly proportional to [S]. Since [S] decreases with time the velocity will also decrease with time. For first order reactions, a constant proportion (not amount) is converted to product per unit time log [S] 0 /[S] t = kt Firstly, calculate k knowing that at the end of 1 minute 98% of the original substrate remains log(100/98) = k(1min) log (1.02) = k min k = 1.98 x 10-2 min -1 Next, calculate [S] t at t = 3 min log(100/[s] t ) = 1.98 x 10-2 min -1 (3 min) 2.303(log log [S] t ) = 5.94 x log [S] t = 5.94 x log [S] t = log [S] t = [S] t = 94.2% of original [S] [S] t = 1 x 10-5 (0.942) = 9.42 x 10-6 M. Therefore, the [P] will be = 5.8% = 1 x 10-5 M (0.058) = 5.8 x 10-7 M. (b) If [S] = 10-6 M the reaction is still first order. The proportion of substrate converted to product would still be 5.6% by 3 min. However, the absolute amount of substrate consumed (and product formed) would obviously be less than in part (a) above. (c) V max can be estimated since we know K M (hence the reaction order) At [S] 0 = 1 x 10-5 M the velocity for the first minute is : v 0 = k[s] v 0 = 0.02 min -1 (1 x 10-5 M) = 2 x 10-7 M min -1 v/v max = [S]/(K M + [S]) 2 x 10-7 M min -1 /V max = 1 x 10-5 M/(2 x 10-3 M) + (1 x 10-5 M) V max = [(2 x x 10-5 )(2 x 10-7 )/(1 x 10-5 )] M min -1 = 4.02 x 10-5 M min -1 (d) V max will be observed at about 100 K M (rule of thumb estimate) therefore 100(K M ) = 100(2 x 10-3 M) = 2 x 10-1 M = 0.2 M (e) At 0.2M [S] 0 the reaction will be zero order [P] = V max (t) = (4.02 x 10-5 M min -1 ) (3 min) = x 10-5 M x 10-5 M/2 x 10-1 M (100) = 6.03 x 10-2 % 0.06% 9. Lineweaver-Burk equation: (1/v 0 ) = K M /V max (1/[S] 0 ) + 1/V max Slope = K M /V max = 2.5 x 10-4 min 9

10 Y intercept = 1/V max = 3.0 x 10-2 M -1 min V max = M min -1 then K M = (33.33 M min -1 )(2.5 x 10-4 min) = 8.33 x 10-3 M 10. (a) v 0 = V max [S]/(K M + [S]) v 0 /V max = [S]/(K M + [S]) v 0 /V max = 1/(10 + 1) = 1/11 = (b) v 0 /V max = 10/(1 + 10) = 10/11 =