Non-competitive Inhibition

1) Pesticide inhibition on enzyme has been reported which caused the enzyme activity to reduce
the collected data with and without inhibition are presented below. Determine the type of
inhibition

S, mmol/cm3 V, I=0.0 V, I=0.20 V, I=0.40 V, I=0.60

0.050 0.015 x 10-3 0.012 x 10-3 0.0098 x 10-3 0.0084 x 10-3
0.10 0.026 x 10-3 0.029 x 10-3 0.017 x 10-3 0015 x 10-3
0.20 0.042 x 10-3 0.033 x 10-3 0.028 x 10-3 0.024 x 10-3
0.40 0.059 x 10-3 0.047 x 10-3 0.039 x 10-3 0.034 x 10-3
0.60 0.069 x 10-3 0.055 x 10-3 0.046 x 10-3 0.039 x 10-3

Required: Type of Inhibition

Solution:
1 𝐾𝑚 1 1
Using Lineweaver-Burk Plot: = +
𝑉 𝑉𝑚𝑎𝑥 𝑆 𝑉𝑚𝑎𝑥

1 1
X=𝑆 Y=𝑉

Lineweaver-Burk Plot
140000

y = 5000.4x + 18983
120000
R² = 0.9915
y = 4387.5x + 14423
100000 R² = 0.9998
y = 3420.2x + 10724
80000 R² = 0.95
1/V

y = 2848.4x + 9763.6
R² = 0.9999
60000

40000
I=0.0
I=0.2
20000
I=0.4
I=0.6
0
0 5 10 15 20 25
1/S

@ I=0.2
Non-competitive Inhibition

Km=0.2917 mmol

Vmax= 1.0242 mmol/s

@ I = 0.4

Km= 0.3189 mmol

Vmax = 9.3249 x 10-5 mmol/s

Noncompetitive
@ I= 0.6 Inhibition

Km = 0.2911mmol

Vmax = 5.2678 x 10-5 mmol/s

@ I=0.8

Km= 0.2834 mmol

Vmax = 5.2678 x 10-5 mmol/s

Non-competitive Inhibition

2) A chemist measured the initial rate of an enzyme-catalyzed reaction in the absence and
presence of inhibitor A and, in a separate procedure, inhibitor B. In each case, the inhibitor’s
concentration was 8.0 mM ð8:0 103 MÞ. The following data were obtained:

S=M v0=M / s No Inhibitor v0=M/s Inhibitor A v0=M/s Inhibitor B

5x 10-4 1.25 x 10-6 5.8 x 10-7 3.8 x 10-7
1 x 10-3 2 x 10-6 1.04 x 10-6 6.3 x 10-7
2.5 x 10-3 3.13 x 10-6 2 x 10-6 1 x 10-6
5 x 10-3 3.85 x 10-6 2.78 x 10-6 1.25 x 10-6
1 x 10-2 4.55 x 10-6 3.57 x 10-6 1.43 x 10-6

Determine the values of KM and Vmax of the enzyme. (b) Determine the type of inhibition imposed by
inhibitors A and B, and calculate the value of KI in each case.

Solution:

 Convert the data to 1/S and 1/Vo

Lineweaver–Burk plots for the three sets of kinetic data.

Linear fit for no inhibition is

Non-competitive Inhibition

1 1
= 302.6 + 1.96𝑥105
𝑉𝑜 𝑆

Vmax=5.1x10-6 M/s

Km=1.5x10-3 M

Linear fit for inhibitor A

1 1
= 757.8 + 2.03 x 105
𝑉𝑜 𝑆

Vmax=4.93 x10-6 M/s

Km=3.73 x10-3 M

𝐾𝑚 [𝐼]
757.8𝑠 = (1 +
𝑉𝑚𝑎𝑥 𝐾𝑖

1.5 𝑥10−3 [8.0 𝑥10−3 ]

= (1 +
5.1 𝑥10−6 𝐾𝑖

Ki = 5.1 x 10-3 M

Competitive
Inhibition

For inhibitor B

1 1
= 1015.3 + 5.95 x 105
𝑉𝑜 𝑆

Vmax= 1.68 x 10-6M/s

Km=1.71 x 10-3 M

𝐾𝑚 [𝐼]
1015.3 𝑠 = (1 + )
𝑉𝑚𝑎𝑥 𝐾𝑖

1.5 𝑥10−3 [8.0 𝑥10−3 ]

= (1 +
5.1 𝑥10−6 𝐾𝑖

Ki=3.3 x 10-3M

Noncompetitive
Inhibition Source: http://www.uscibooks.com/changten.pdf
Non-competitive Inhibition

3) The following table shows the rate of reaction of substrate to product in the presence of enzyme;
v (mol/sec) under different conditions;

1. Reaction 1: Reaction of substrate with enzyme to form product in the absence of inhibitor.
2. Reactions 2 and 3: Reaction takes place in the presence of two different inhibitors, each with
10mM concentration.

v, reaction 2 v, reaction 3
v, reaction 1
[S] mM (inhibitor A) (inhibitor B)
(μ mol/sec)
(μ mol/sec) (μ mol/sec)
1 2.5 1.17 0.77
2 4.0 2.1 1.25
5 6.3 4.0 2.0
10 7.6 5.7 2.5
20 9.0 7.2 2.86

Determine the values of KM and Vmax of the enzyme. (b) Determine the type of inhibition
imposed by inhibitors A and B, and calculate the value of KI in each case.

Solution:
1 𝐾𝑚 1 1
Using Lineweaver-Burk Plot : 𝑉 = 𝑉𝑚𝑎𝑥 𝑆 + 𝑉𝑚𝑎𝑥

Lineweaver-Burk Plot y = 0.9989x + 0.3001

R² = 1
1.4
y = 0.7541x + 0.1
1.2 R² = 1
1 y = 0.3017x + 0.0987
R² = 0.9997
0.8
1/V

0.6 0
0.4 A
0.2 B
0 Linear (0)
0 0.2 0.4 0.6 0.8 1 1.2
Linear (A)
1/S
Linear (B)
Non-competitive Inhibition

No Inhibitor:

Km = 3.3 mM, Vmax = 10 mmol/sec

Inhibitor A

Km = 7.7 mM Vmax = 10 mmol/sec Competitive

Inhibition
Ki=7.52 mM

Inhibitor B

Km = 3.3 mM, Vmax= 3.33 mmo l / s e c

Noncompetitive
Ki=5 mM Inhibition

Source: http://chemistry.tutorvista.com/biochemistry/enzyme-kinetics.html