2021-1 Enzyme Engineering / Problem set #3  Due: May 6

Solve the following problems. In case you need plots, use a 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 (nM/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 10-5 M ?

2. 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 (Hints: initial velocity can be determined by plotting time vs. A300 for each substrate (NAD+) concentration. Absorbance directly reflects reaction rate).

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

(1) What is the inhibition type of this reaction?

(2) Calculate the degree of inhibition.