2019-1 Enzyme Engineering   Homework 2 Due on May 22, 2019

Solve the following problems. In case you need plots, use computer graphic software such as Excel or Sigma Plot. Clearly indicate the units of answer.

1. The following data were recorded for the enzyme-catalyzed reaction S→P.

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S (M)                     V (mM/min)

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6.25 x 10-6                    15

7.50 x 10-5                    56.25

1.00 x 10-4                    60

1.00 x 10-3                    74.9

1.00 x 10-2                    75

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1) Estimate Km and Vmax.

2) What would V be at S=2.5 x 10-5M ?

2. Determine the Michaelis-Menten equation for the following reaction:

urea + urease = [urea․urease] → 2 NH3 + CO2 + urease

The rate of equation is given as a function of urea concentration in the               following table.

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Curea (kmol/m3)              V (kmol/m3․s)

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0.2                        1.08

0.02                       0.55

0.01                       0.38

0.005                      0.2

0.002                      0.09

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3. We can directly determine kinetic parameters (Vmax and Km) in terms of reaction time (t) versus substrate concentration (S0 and S). Calculate the Vmax and Km using the following data. You may use a semi-log plots for solving this problem (Hint: modify the integrated Michaelis-Menten equation).

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Time (min)           S (mol/liter)

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0                     0.1

10                     0.089

20                     0.079

50                     0.053

100                     0.025

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4. Malate dehydrogenase catalyses the reaction:

(S)-malate + NAD+ = oxaloacetate + NADH+H+

The rate of the forward reaction was investigated in the presence of saturating concentrations of malate and a fixed concentration of enzyme. The following results were obtained. Calculate the kinetic parameters (Km and Vmax) in this reaction.

NAD+ concentration (mmol/l)	Absorbance at 300 nm (A300) at time t (min) x 102
	t=0.5	t=1	t=1.5	t=2	t=2.5	t=3
1.5	3.3	5.6	7.9	10.2	12.2	13.8
2	3.6	6.3	8.9	11.6	13.8	15.4
2.5	4.0	6.9	9.9	12.8	15.0	16.8
3.33	4.3	7.5	10.8	14.0	16.3	17.5
5	4.7	8.4	12.1	15.8	17.7	18.4
10	5.2	9.5	13.7	18.0	19.2	20.0

[Hints] Determine the reaction velocity for each cosubstrate concentration (NAD+). (Example) For 1.5 nmol/l NAD+

A300

Slope=reaction                   velocity

time (min)