Group 3

Callueng, Jomhel B. Mora, Roxan B.

Lozano, John Harvey S. Pasion, Cherilyn S.

2.5 Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholme (substrate) by
dog serum (source of enzyme) and obtained the following data:

Substrate Concentration Initial Reaction Rate

mol/L mol/L min
0.0032 0.111
0.0049 0.148
0.0062 0.143
0.0080 0.166
0.0095 0.200

Evaluate the Michaelis – Menten kinetic parameters by employing (a) the Langmuir plot, (b) the
Lineweaver – Burk plot, (c) the Eadie – Hofstee plot, and (d) non – linear regression procedure.

Solution:

a. Langmuir Equation

Cs r Cs/r
0.0032 0.111 0.028829
0.0049 0.148 0.033108
0.0062 0.143 0.043357
0.008 0.166 0.048193
0.0095 0.2 0.0475
 Langmuir Plot
0.06

0.05

0.04
Cs/r

0.03 y = 3.3133x + 0.0191

R² = 0.8837
0.02

0.01

0
0 0.002 0.004 0.006 0.008 0.01
Cs

Vmax = 0.301813901 mol/L min

Km = 0.005764645509 mol/L

b. Lineweaver – Burk equation

Cs 1/Cs V 1/V
0.0032 312.5 0.111 9.009009
0.0049 204.0816 0.148 6.756757
0.0062 161.2903 0.143 6.993007
0.008 125 0.166 6.024096
0.0095 105.2632 0.2 5

Lineweaver-Burk Plot
10
9
8
7
6 y = 0.0172x + 3.6342
1/V

5 R² = 0.9146
4
3
2
1
0
0 50 100 150 200 250 300 350
1/Cs
 1 1 K 1
  m
V Vmax Vmax C s

Vmax = 0.2751631224 mol/L min

Km = 0.004732816026 mol/L

c. Eadie – Hofstee equation

Cs r/Cs r
0.0032 34.6875 0.111
0.0049 30.20408 0.148
0.0062 23.06452 0.143
0.008 20.75 0.166
0.0095 21.05263 0.2

Eadie Hofstee Plot

0.22

0.17

0.12
r

0.07 y = -0.0043x + 0.2645

R² = 0.6584

0.02

15 20 25 30 35
-0.03
r/Cs

r
r  K m  rmax
Cs

Vmax = 0.2645 mol/Lmin

Km = 0.0043 mol/Lmin
2.15 Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholme (substrate) by
dog serum (source of enzyme in the absence and presence of prostigmine (inhibitor),
mol
1.5  10 7 and obtained the following data:
L

Initial Reaction Rate (mol/L min)

Substrate Concentartion Absence of Presence of

(mol/L) Prostigmine Prostigmine
0.0032 0.111 0.059
0.0049 0.148 0.071
0.0062 0.143 0.091
0.008 0.166 0.111
0.0095 0.2 0.125

a. Is prostigimine competitive or non-competitive inhibitor?

b. Evaluate the Michaelis-Menten Kinetic parameters in the presence of inhibitor by
employing the Langmuir Plot
Solution:
a.

Substrate
Absence of Presence of
Concentartion Cs/R Cs/R
Prostigmine Prostigmine
(mol/L)
0.0032 0.111 0.028828829 0.059 0.054237
0.0049 0.148 0.033108108 0.071 0.069014
0.0062 0.143 0.043356643 0.091 0.068132
0.008 0.166 0.048192771 0.111 0.072072
0.0095 0.2 0.0475 0.125 0.076
Answer:

Langmuir Plot of Hydrolysis of Acetylchline

0.09 y = 2.9883x + 0.0489
0.08 R² = 0.8137
0.07
0.06
0.05
Cs/R

0.04
0.03 y = 3.3133x + 0.0191
R² = 0.8837
0.02
0.01
0
-0.02 -0.015 -0.01 -0.005 -0.01 0 0.005 0.01 0.015

Cs
with inhibitor without inhibitor

Based on the graph shown, the Km obtained from with and without the presence of inhibitor
are 0.01636819 and 5.76464552  10 3 respectively, are not equal. Therefore, it is a competitive
inhibitor.

b. Langmuir with the presence of Prostigmine

y  2.9883x  0.0489
CS 1 KM
 CS 
r rmax rmax
1
 2.9883
rmax
mol
rmax  0.334638423
L  min
KM
 0.0489
rmax
K M  0.334638423  0.0489
mol
K M  0.016363819
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.,
jBiol.Chem.,233, 159, 1958):

Substrate Concentration Initial Reaction Rate

μmol/L μmol/L min
6.7 0.30
3.5 0.25
1.7 0.16

Calculate the Michaelis – Menten constants of the above reaction.

Solution:

a. Langmuir Equation

Cs Cs/r r
6.7 22.33333 0.3
3.5 14 0.25
1.7 10.625 0.16

Langmuir Plot
25

20

15
Cs/R

10 y = 2.3722x + 6.2429
R² = 0.9937

0
0 1 2 3 4 5 6 7 8
Cs

Vmax = 0.4215496164 μmol/L min

Km = 2.6316921 μmol/L
b. Lineweaver Burk Equation

Cs V 1/Cs 1/V
6.7 0.3 0.149254 3.333333
3.5 0.25 0.285714 4
1.7 0.16 0.588235 6.25

Lineweaver-Burk Plot
7

4
1/V

y = 6.7758x + 2.2168
3 R² = 0.9922
2

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

1 1 K 1
  m
V Vmax Vmax C s

Vmax = 0.4511006857 μmol/L min

Km = 3.056568026 μmol/L

c. Eadie – Hofstee

r/Cs R Cs
0.044776 0.3 6.7
0.071429 0.25 3.5
0.094118 0.16 1.7
 Eadie Hofstee Plot
0.35

0.3

0.25

0.2
r

0.15
y = -2.8096x + 0.4336
0.1 R² = 0.9566

0.05

0
0 0.02 0.04 0.06 0.08 0.1
r/Cs

r
r  K m  rmax
Cs

Vmax = 0.4336 μmol/L min

Km = 2.8096 μmol/L

The linear transform of Michaelis-Menten equation with the highest coefficient of

determination is Langmuir plot with the value of R2=0.9937. This means that the value of Km and
Rmax is adapted from the plot.

Final answer:

Michaelis-Menten Plot
0.35
0.3
0.25
0.2
rp

0.15
0.1
0.05
0
0 1 2 3 4 5 6 7 8
Cs
 Vmax C s
V
K m  Cs

Vmax = 0.4215496164 μmol/L min

Km = 2.6316921 μmol/L

2.19b When the inhibitor was added, the initial reaction rate was decreased as follows:

Substrate Concentration Inhibitor Initial Reaction Rate

μmol/L μmol/L μmol/L min
6.7 146 0.11
3.5 146 0.08
1.7 146 0.06
Is this competitive inhibition or noncompetitive inhibition? Justify your answer by showing the
effect of the inhibitor graphically.

Solution:

a. Langmuir Equation

Cs Cs/r r
6.7 60.9091 0.11
3.5 43.75 0.08
1.7 28.3333 0.06

Langmuir Plot of Enzyamtic Cleavage of

Deoxyguanosine Triphospate
70
60 y = 6.3809x + 19.02
50 R² = 0.9833

40 y = 2.3722x + 6.2429
Cs/r

30 R² = 0.9937
20
10
0
-4 -2 -10 0 2 4 6 8
Cs

Linear (Without Inhibitor) Linear (With Inhibitor)

 Km Vmax
(μmol/L) (μmol/L min)
Without Inhibitor 2.6316921 0.4215496164
With Inhibitor 0.335483701 0.052576236

b. Lineweaver-Burk Equation

Cs V 1/Cs 1/V
6.7 0.11 0.149254 9.090909
3.5 0.08 0.285714 12.5
1.7 0.06 0.588235 16.66667

Lineweaver-Burk Plot of Enzyamtic Cleavage

of Deoxyguanosine Triphospate
20
y = 16.68x + 7.0637
15 R² = 0.9754

10
1/V

5
y = 6.7758x + 2.2168
0 R² = 0.9922
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
-5
1/Cs

Linear (With Inhibitor) Linear (Without Inhibitor)

1 1 K 1
  m
V Vmax Vmax C s

Vmax = 0.1415688662 μmol/L min

Km = 2.3613686878 μmol/L
 Km Vmax
(μmol/L) (μmol/L min)
Without Inhibitor 3.056568026 0.4511006857
With Inhibitor 2.3613686878 0.1415688662

Based on the graphs, the values of Km are almost the same and the values of the Vmax for
both with and without inhibitor are not equal. Hence, we can consider that the enzymatic cleavage
of Deoxyguanosine Triphosphate is a noncompetitive inhibition.

2.20 The effect of an inhibitor on an enzyme reaction was studied by measuring the initial rates
at three different initial inhibitor concentrations. The obtained Michaelis-Menten kinetic
parameters are as follows:

Inhibitor rmax KM
µmol/L µmol/L min µmol/L
0 0.70 5
2 0.20 5
4 0.11 5
6 0.08 5
a. Write the kinetic model for this enzyme reaction.
b. Derive the rate equation. State your assumptions for any simplification of the equation.
c. Estimate the value of inhibition kinetic parameter.

Answer:

a. Since the given has constant KM shown in the table given table, then the enzyme reaction
is non-competitive inhibition reaction.
The kinetic model would be:
 K1

E +S ⇔
K
ES
2

K3

E+I ⇔
K4
EI
K5

EI + S ⇔
K6
EIS
K7

ES + I ⇔
K
ESI
8

K9

ES → E + P
b. Assumptions:
 The dissociation constant for the first equilibrium reaction is the same as that of
the third equilibrium reaction.
 The dissociation constant for the second equilibrium reaction is the same as that
of the fourth equilibrium equation

The two equilibrium reactions,

k2 k
 K S  6  K IS
k1 k5

k4 k
 K I  8  K SI
k3 k7

If the slower reaction, the product formation step, determines the rate of the reaction according
to Michaelis-Menten assumption, the rate can be expressed as:

rp  k9 ES  (1)

The enzyme balance gives

E0   E   ES   EI   ESI  (2)

Divide (1) by (2),

rp k9 ES 
 (3)
E0  E   ES   EI   ESI 
Applied Law of Mass Action
 K 2 E S  E S 
KS    ES   (4)
K1 ES  KS

K 4 E I  E I 
KI    EI   (5)
K3 EI  KI

K 8 ES I  ES I 

KS    ESI   (6)
K7 ESI  KI

Substitute (4), (5), (6) into (3),

k9
E S 
rp K

E 0  E   E S   E I   ES I 
KS KI KI

k9
E S 
rp K

E 0  E   E S   E I   E S I 
KS KI KS KI

Elininate [E],

S 
rp KS

EO  k9 1
S   I   S I 
KS KI KS KI

Substitute rpmax  E 0  k 9

S 
rp KS

rpmax 1
S   I   S I 
KS KI KS KI

Multiply numerator and denominator by K S p

 rp

S 
K S I  S I 
K S  S  
r pmax

KI KI

Rearranging

rp

S 
K I  S I 
rpmax KS  S  S  
KI KI

rp

S 
rpmax 
K S 1 
I    S  1  I  
  K 
 KI   I 