Problem Set

In
Introduction to Biotechnology

Submitted by:

Princcess Janine B. Catral BSChE IV

Submitted to:

Engr. Monico U. Tenedor

 1. Eadie measured the reaction rate of hydrolysis of acetylcholine (substrate) by dry serum (source of
enzyme) and obtained the following data:

Substrate Concentration, mol/L Initial Reaction Rate, mol/L min

0.0032 0.111
0.0049 0.148
0.0062 0.143
0.008 0.166
0.0095 0.200
Evaluate Michaelis Menten parameters by employing: a) Langmuir plot b) Lineweaver-Burke plot c)
Eadie Hofstee plot d) non-linear Michaelis Menten plot

The following calculations were made:

Substrate Concentration, Initial Reaction Rate, mol/L 1/Cs 1/V Cs/V V/Cs
mol/L min
0.0032 0.111 312.5 9.009009 0.028829 34.6875
0.0049 0.148 204.0816 6.756757 0.033108 30.20408163
0.0062 0.143 161.2903 6.993007 0.043357 23.06451613
0.008 0.166 125 6.024096 0.048193 20.75
0.0095 0.2 105.2632 5 0.0475 21.05263158

a. Langmuir plot

Langmuir plot Langmuir plot

(-) 1/Vmax 3.313 0.12
Km/Vmax 0.019
0.1 y = 3.3133x + 0.0191
Vmax 0.301841232
0.08
Km 0.005734983
0.06
Cs/V 0.04
0.02
0
-0.015 -0.01 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03
-0.02
-0.04
Cs
b. Lineweaver-Burke plot

Lineweaver- Burke Plot Lineweaver- Burke plot

Km/Vmax 0.017
20
1/Vmax 3.634
Vmax 0.275178866 y = 0.0172x + 3.6342
15
Km 0.004678041
10

1/V 5

0
-600 -400 -200 0 200 400 600 800
-5

-10
1/Cs

c. Eadie-Hofstee Plot

Eadie Hofstee plot

(-) Km -0.004
Vmax 0.264 Eadie-Hofstee plot
Km 0.004 0.5

0.4

0.3
y = -0.0043x + 0.2645
0.2
V
0.1

0
-40 -20 0 20 40 60 80 100
-0.1

-0.2
V/Cs
d. Non-Linear Michaelis Menten approach

Non-Linear Michaelis Menten plot

0.45
0.4
0.35 y = 0.0718ln(x) + 0.5216
0.3
0.25
V
0.2
0.15
0.1
0.05
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18
Cs
2. Eadie measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by dry serum (source
of enzyme) in the absence and presence of prostigmine (inhibitor), 1.5 x 10-7 mol/L and obtained the
following data:

Substrate concentration, mol/L Initial Reaction Rate mol/Lmin

Absence of Prostigmine Presence of Prostigmine
0.0032 0.111 0.059
0.0049 0.148 0.071
0.0062 0.143 0.091
0.0080 0.166 0.111
0.0095 0.200 0.125

A) Is prostigmine competitive or non-competitive inhibitor? Show graphically

B) Evaluate Michaelis-Menten kinetic parameters in the presence of inhibitor by employing the
Lineweaver-Burke plot.

The following data were obtained:

Substrate Initial Reaction Rate 1/Cs 1/V (no 1/V (with V/S (no V/S (with
concentration, mol/Lmin inhibitor) inhibitor) inhibitor inhibitor)
mol/L )
Absence of Presence of
Prostigmine Prostigmine
0.0032 0.111 0.059 312.5 9.00900 16.94915 34.6875 18.4375
0.0049 0.148 0.071 204.0 6.75675 14.08450 30.2040 14.48979
81633
0.0062 0.143 0.091 161.2 6.99300 10.98901 23.0645 14.6774
0.008 0.166 0.111 125 6.02409 9.009009 20.75 13.875
6386 009
0.0095 0.2 0.125 105.2 5 8 21.0526 13.15789
63158 3158 474
Using Lineweaver-Burke plot, it shows that prostigmine is a competitive inhibitor since both plots have
the same y-intercept (1/Vmax):

Lineweaver-Burke Plot
50
y = 0.0448x + 3.634
40
30
20
1/V 10 Absence of
y = 0.0172x + 3.634
0 Prostigmine
-600 -400 -200 0 200 400 600 800 1000
-10
-20
-30
1/Cs

Michaelis Menten Parameters Absence of Prostigmine Presence of Prostigmine

Km/Vmax 0.017 0.044
1/Vmax 3.634 3.634
Vmax 0.275178866 0.275178866
Km 0.004678041 0.01210787

In comparison with the Eadie-Hofstee plot, it also shows that prostigmine is a competitive inhibitor

Eadie Hofstee plot

0.8

0.6

0.4

0.2 y = -0.0043x + 0.2645

No Prostigmine
V
0 With Prostigmine
-40 -20 0 20 40 60 80
-0.2

-0.4 y = -0.0116x + 0.264

-0.6
V/Cs
Michaelis Menten Parameters Absence of Prostigmine Presence of Prostigmine
(-)Km (-) 0.017 (-)0.043
Vmax 0.264 0.264
Km 0.017 0.043
 3. The initial rate of reaction for the enzymatic cleavage of deoxygranosine triphosphate was measured
as a function of initial substrate concentration as follows:
Substrate concentration, mol/L Initial reaction rate µmol/Lmin
6.7 0.30
3.5 0.25
1.7 0.16
Calculate the Vmax and Km for the reaction. When the inhibitor was added, the initial reaction rate was
decreased as follows:
Substrate, mol/L Inhibitor, µmol/L Initial Reaction Rate µmol/Lmin
6.7 146 0.11
3.5 146 0.08
1.7 146 0.06
Is the inhibitor competitive or non-competitive?

The following data were calculated:

Cs Initial reaction rate Initial Reaction Rate 1/Cs 1/V (no 1/V (with V/Cs (no V/Cs (with
mol µmol/Lmin (without µmol/Lmin (with inhibitor) inhibitor) inhibitor) inhibitor
/L inhibitor) inhibitor)
6.7 0.3 0.11 0.14 3.333333 9.090909 0.044776 0.0164179
9254 333 091 119 1
3.5 0.25 0.08 0.28 4 12.5 0.071428 0.0228571
5714 571 43
1.7 0.16 0.06 0.58 6.25 16.66666 0.094117 0.0352941
8235 667 647 18

Using Lineweaver-Burke plot:

Lineweaver-Burke Plot
35
30 y = 16.68x + 7.0637
25
20
Lineweaver-Burke Plot No
15
1/V y = 6.7758x + 2.2168 inhibitor
10
Lineweaver-Burke Plot With
5 inhibitor
0
-1 -0.5 -5 0 0.5 1 1.5
-10
1/Cs
 Michaelis-Menten Parameters No inhibitor With inhibitor
Km/Vmax 6.775 16.68
1/Vmax 2.216 7.063
Vmax 0.451263538 0.141582897
Km 3.057310469 2.361602718

Using the Eadie-Hofstee Plot

Eadie-Hofstee Plot
1.2
1
0.8
0.6
0.4 No inhibitor
0.2 Witn inhibitor
y = -1.0166x + 0.1546
V 0
-0.4 -0.2 -0.2 0 0.2 0.4 0.6
-0.4
-0.6 y = -2.8128x + 0.154
-0.8
-1
V/Cs

Michaelis-Menten Parameters No inhibitor With inhibitor

(-) Km (-) 1.016 (-) 2.812
Vmax 0.154 0.154
Km 1.016 2.812

The inhibitor is competitive.