DEPARTMENT OF CHEMICAL ENGINEERING

N ATIONAL I NSTITUTE OF T ECHNOLOGY

WINTER SEMESTER 2018-19
CH4024 BIOCHEMICAL ENGINEERING

Problem Set 2: Complex Enzyme Kinetics

1 The kinetics of allosteric enzymes can be represented using the overall reaction:

E + nS −
)−
−*
− ESn −−→ E + nP

Derive Hill equation using rapid equilibrium approach.

2 Derive a rate equation for the following partially competitive inhibition using the Michaelis-Menten approach.

E+S −
)−
−*
− ES
E+I −
)−
−*
− EI
ES + I −
)−
−*
− EIS
−−*
EI + S )−− EIS
ES −−→ E + P
EIS −−→ EI + P

3 The initial rate of reaction for the enzymatic cleavage of deoxyguanosine triphosphate was measured as a function of
initial substrate concentration as follows:

Substrate Concentration Initial Reaction Rate in

in µmol/L µmol/L min
6.7 0.30
3.5 0.25
1.7 0.16

(a) Calculate the Michaelis-Menten constants of the above reaction.

(b) When the inhibitor was added, the initial reaction rate was decreased as follows:

Substrate Concentration Inhibitor Concentration Initial Reaction Rate in

in µmol/L in µmol/L µmol/L min
6.7 146 0.11
3.5 146 0.08
1.7 146 0.06

Determine the type of inhibition.

4 During a test of kinetics of an enzyme-catalyzed reaction, the following data were recorded:

E0 (g/L) T (◦ C) I (mmol/mL) S (mmol/mL) v (mmol/mL-min)

1.6 30 0 0.1 2.63
1.6 30 0 0.033 1.92
1.6 30 0 0.02 1.47
1.6 30 0 0.01 0.96
1.6 30 0 0.005 0.56
1.6 49.6 0 0.1 5.13
1.6 49.6 0 0.033 3.70
1.6 49.6 0 0.01 1.89
1.6 49.6 0 0.0067 1.43

CH4024 BIOCHEMICAL ENGINEERING Problem Set - 2 Page 1 of 2

 1.6 49.6 0 0.005 1.11
0.92 30 0 0.1 1.64
0.92 30 0 0.02 0.90
0.92 30 0 0.01 0.58
0.92 30 0.6 0.1 1.33
0.92 30 0.6 0.033 0.80
0.92 30 0.6 0.02 0.57

(a) Determine the Michaelis-Menten constant for the reaction with no inhibitor present at 30◦ C and at 49.6◦ C.
(b) Determine the maximum velocity of the uninhibited reaction at 30◦ C and an enzyme concentration 1.6 g/L.
(c) Determine the KI for the inhibitor at 30◦ C and decide what type of inhibitor is being used.

5 The effect of an inhibitor on an enzyme reaction was studied by measuring the initial rates at three different initial
inhibitor concentrations. The obtained Michelis-Menten kinetic parameters are as follows:

Inhibitor Concentration vmax in µmol/L min KM in µmol/L

in µmol/L
0 0.70 5
2 0.20 5
4 0.11 5
6 0.08 5

(a) Determine the type of inhibition.

(b) Write the kinetic model for this enzyme reaction.
(c) Estimate the value of inhibition kinetic parameter.

6 An inhibitor (I) is added to the enzymatic reaction at a level of 1.0 g/L. The following data were obtained for Km =9.2g
S/L.

v in gL−1 min 0.909 0.658 0.493 0.40 0.333 0.289 0.227

S in gL−1 20 10 6.67 5 4 3.33 2.5

(a) Is the inhibitor competitive or non-competitive?

(b) Find KI .

7 An enzyme ATPase has a molecular weight 5 × 104 daltons, a Km value of 10−4 M, and a k2 value of 104 molecules
ATP/min molecule enzyme at 37◦ C. The reaction catalyzed is the following:
ATPase
ATP −−−−→ ADP + Pi

which can also be represented as

k1 k
−−
E+S )−* 2
− ES −−→ E + P
k−1

where S is ATP. The enzyme at this temperature is unstable. The enzyme inactivation kinetics is first order:

E = E0 e−kd t

where E0 is the initial enzyme concentration and kd =0.1 min−1 . In an experiment with a partially pure enzyme
preparation, 10µg of total crude protein (containing enzyme) is added to a 1 ml reaction mixture containing 0.02 M
ATP and incubated at 37◦ C. After 12 hours the reaction ends and the inorganic phosphate (Pi ) concentration is found
to be 0.002 M, which was initially zero. What fraction of the crude protein preparation was the enzyme?
Hint: Since [S]  Km , the reaction rate can be represented by dP
dt = k2 [E]

End

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