Orpilla, Jerryson M.

BSChE-5

2.15. Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by
dog 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 Initial Reaction Rate Rate (mol/L-min)

Cs/r Cs/r
Concentration, Cs Absence of Presence of
(Uninhibited) (Inhibited)
(mol/L) Prostigmine Prostigmine
0.0032 0.111 0.059 0.028828829 0.05423729
0.0049 0.148 0.071 0.033108108 0.06901408
0.0062 0.143 0.091 0.043356643 0.06813187
0.008 0.166 0.111 0.048192771 0.07207207
0.0095 0.2 0.125 0.0475 0.076

a. Is prostigmine competitive or noncompetitive inhibitor?

b. Evaluate the Michaelis-Menten kinetic parameters in the presence of inhibitor by employing

the Langmuir Plot.

Answers:

Langmuir Plot of Hydrolysis of Acetylcholine

0.09

0.08 y = 2.9883x + 0.0489

0.07

0.06

0.05 y = 3.3133x + 0.0191

Cs/r 0.04

0.03

0.02
-KMI -KM
0.01

0
-0.02 -0.015 -0.01 -0.005 0 0.005 0.01 0.015
-0.01
Cs

Without Inhibitor With Inhibitor

a. Based from the graph, the values of Km with and without the presence of inhibitor is not
equal. Therefore, prostigmine is a competitive inhibitor.

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Orpilla, Jerryson M. BSChE-5

b. Langmuir Equation (With inhibitor):

Cs 1 K
 Cs  m
r rmax rmax

y  2.9883x  0.0489

1
 2.9883
rmax

mol
rmax  0.3346
L  min

K mi
 0.0489
rmax

mol
K mi  0.01636
L

2.19. The initial rate of reaction for the enzymatic cleavage of deoxyguanosine triphosphate was
measured as a function of initial substrate concentration as follows (Kornberg et al., J.BioI.Chern.,
233, 159, 1958):

Substrate Concentration Initial Reaction Rate (µmol/L-

1/Cs 1/r
(µmol/L) min)
6.7 0.3 0.149254 3.333333
3.5 0.25 0.285714 4
1.7 0.16 0.588235 6.25
a. Calculate the Michaelis-Menten constants of the above reaction.

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

Initial Reaction
Substrate Concentration
Inhibitor Rate (µmol/L- 1/r
(µmol/L)
(µmol/L) min)
6.7 146 0.11 9.090909
3.5 146 0.08 12.5
1.7 146 0.06 16.66667

Is this competitive inhibition or noncompetitive inhibition? Justify your answer by showing the
effect of the inhibitor graphically. [Contributed by Professor Gary F. Bennett, The University of
Toledo, Toledo, OH]

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Orpilla, Jerryson M. BSChE-5

Answers:

Lineweaver-Burk Plot of Enzymatic Cleave

of Deoxyguanosine Triphosphate
y = 6.7758x + 2.2168
7
6
5
4
1/r

3
2
1
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
1/Cs

a. Lineweaver-Burk Equation

1 Km 1 1
 
r rmax Cs rmax

y  6.7758 x  2.2168

1
 2.2168
rmax

mol
rmax  0.451
L  min

Km
 6.7758
rmax

mol
K m  3.0566
L

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Orpilla, Jerryson M. BSChE-5

b. Based from the graph, the values of Km with and without inhibitor are almost the same.
Moreover, the y-intercepts, rmax of the Lineweaver-Burk for both inhibited and uninhibited are
not equal. Therefore, it is a noncompetitive inhibition.

Lineweaver-Burk Plot of Enzymatic Cleavage of

Deoxyguanosine Triphosphate
18
16 y = 16.68x + 7.0637
14
12
10
y = 6.7758x + 2.2168
1/r 8
6
4
2
0
-0.6 -0.4 -0.2 -2 0 0.2 0.4 0.6 0.8
1/Cs

Without inhibitor WIth inhibitor

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