CLL 277

Tutorial 1- Enzyme Kinetics

Basics to revise before solving:
A)Michaelis-Menten enzyme kinetics
B) Enzyme inhibition
Q1. For a single substrate enzyme catalyzed reaction given as:
K1

K2

E + S ES E+P
K-1

a) Derive the Michaelis-Menten equation for enzyme kinetics for S o

>> Eo. Where Eo and So represent the initial enzyme and substrate
concentration respectively.
b) Derive the expression for Km and Vmax in terms of the reaction
rate constant(s) and initial enzyme concentration (Eo).
c) Determine the fraction of Vmax that would be obtained at the
following substrate concentration [S]: 1/2 Km, 2Km and 10 Km.
Q2. The following data have been obtained for two different initial
enzyme concentration for an enzyme catalyzed reaction.
V (for [Eo] = 0.015 g/L)
(g/L min)
1.14
0.87
0.70
0.59
0.50
0.44
0.39
0.35

[S]
(g/L)
20
10
6.7
5.0
4.0
3.3
2.9
2.5

V (for [Eo] = 0.00875 g/L)

(g/L min)
0.67
0.51
0.41
0.34
0.29

a) Find Km
b) Find Vmax for Eo = 0.015 g/L
c) Find Vmax for Eo = 0.00875 g/L
d) Find K2 (rate constant for the enzyme catalyzed reaction limiting
step)
Q3. An enzyme is discovered that catalyzes the chemical reaction
Glucose Glucose 6 phosphate
A team of motivated researchers sets out to study the enzyme which they
call hexokinase. They find that the Kcat for hexokinase is 600 S-1.
a) Kcat stands for enzyme turnover number. What does turnover
number for an enzyme represent? Write the Michaelis-Menten
equation with respect to the turnover number.
b) When [Eo] is 25 nM and [Glucose] is 40 M, the reaction velocity
V is 9.6 M/S. Calculate Km for the substrate Glucose.
c) In a separate hexokinase experiment using [Eo]= 10 nM, the
reaction rate V is measured as 3 M/S. Calculate substrate
concentration [S] used in the reaction.

Q4.Write the expressions for the each of the different types of reversible
inhibition: a) Competitive Inhibition
b) Non-competitive Inhibition
c) Uncompetitive Inhibition
Also describe briefly the qualitative difference between the three in
terms of increase or decrease in the value of Km and Vmax. Also depict
the same graphically with the help of Lineweaver-Burk plots.

Q5. Malonate, a competitive inhibitor of succinate dehydrogenase, was

found to cause 99% inhibition of the enzymes activity.
(a) If the succinate (substrate) concentration for the enzyme was 3.5 x
10-5 M and the Km for this succinate is 4.4 x 10-6 M, what was the initial
Malonate (Inhibitor) concentration (Ki = 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?