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

8
y = 0.0172x + 3.6342
1/V

7 R² = 0.9146

4
100 150 200 250 300
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
2.19 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