TUTORIAL 1

1. Look up recent literature on the different kinds of enzymes that are required by the
human body to carry out vital operations. List and explain the function of any five
such enzymes and the quantities in which they are required by the body.
2. What is the Human Genome project? Explain the advances made in it, and the
challenges still remaining, in 80-100 words.

3. What are the equations and conditions of existence of:i.

Non-competitive inhibition
ii.
Competitive Inhibition
iii.
Uncompetitive Inhibition
4. i. Derive the expression for the product (P) concentration in the following reaction:

by using the pseudo steady state approximation.

ii. Show that the results of the previous problems can be used to derive the Michaelis-Menten
enzyme kinetics model:

[]

(E = enzyme, S = substrate, and P = product) with = k0 [E]0,

k0 = k[S] / (KM + [S]) and KM = (k + k+)/k+. Note that the substrate is in excess compared to
the enzyme and the concentration of the enzyme is conserved in the reaction.

5. The kinetics of an enzyme were analyzed in the presence and absence of inhibitors A
and B
[S]
mM
1.0
2.0
5.0
10.0
20.0

No inhibitor
43
68
105
128
144

Vo (nmoles/min}
5 mM A
30
50
86
113
134

0.1 mM B
26
41
64
77
88

A. What type of inhibitors are A and B: Competitive or Non-competitive?

(Use your graphs to answer this question)

B. In addition to calculating Km and Vmax in absence of inhibitors calculate Ki

constants for the inhibitors A and B.
Graph for 5:-

Linweaver Burke Plot

0.045
0.04

y = 0.0285x + 0.01

0.035

y = 0.0273x + 0.0062

No Inhibitor

1/V0

0.03
0.025

5 mM A

y = 0.0172x + 0.0061

0.02

0.1 mM B

0.015

Linear (No Inhibitor)

0.01

Linear (5 mM A)

0.005

Linear (0.1 mM B)

0
0

0.2

0.4

0.6

0.8

1.2

1/[S]0

6. You have purified a new enzyme and want to determine its kinetic properties in the
presence and absence of two inhibitors (I-1 and I-2). The experimental conditions
are:a. 1ml reaction volume, pH = 7.5, 0.1M KCl
b. The substrate is varied between 1 and 20mM
c. amount of enzyme added = 0.2 microgram of mol wt. 100,000 g/mol
d. incubation time of 5min at 25 C
The following data were obtained:[S] mM
1
2
5
10
20

Velocity of reaction nano mol/min

No inhibitor
5mM I-1
10mM I-2
2.5
1.17
0.77
4
2.1
1.25
6.3
4
2
7.6
5.7
2.5
9
7.2
2.86

A. Are the conditions outlined here compatible with the Michaelis Menten model?
B. Determine the Km and Vmax from the given data. Estimate turnover number from Vmax
C. What would be a suitable substrate concentration to use as an assay for this enzyme in
a crude cell extract?