Biochemical Engineering

Problem 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 Plot
Table 1. Langmuir Plot Data for the Hydrolysis of Acetylcholme by Dog Serum.
Cs, mol/L Cs/r,min
0.0032 0.028829
0.0049 0.033108
0.0062 0.043357
0.008 0.048193
0.0095 0.0475
Linear Form
Figure 1. Langmuir Plot, Linear Form
Langmuir Plot for the Hydrolysis of Acetylcholme by
Dog Serum
0.06 y = 3.3133x + 0.0191
R² = 0.8837
0.05

0.04
Cs/r, min

0.03

0.02

0.01

0
0 0.002 0.004 0.006 0.008 0.01
Cs, mol/L

Polynomial Form, n=3

Figure 2. Langmuir Plot, Polynomial Form (n=3)
 Langmuir Plot for the Hydrolysis of Acetylcholme by
Dog Serum
0.06
y = -255302x3 + 4399.3x2 - 19.494x + 0.0543
0.05 R² = 0.9823
0.04
Cs/r

0.03

0.02

0.01

0
0 0.002 0.004 0.006 0.008 0.01
Cs

Polynomial Form, n>3

Figure 3. Langmuir Plot, Polynomial Form (n>3)
Langmuir Plot for the Hydrolysis of Acetylcholme by
Dog Serum
0.06
y = 1E+08x4 - 4E+06x3 + 37266x2 - 146.66x + 0.2275
0.05 R² = 1
0.04
Cs/r

0.03

0.02

0.01

0
0 0.002 0.004 0.006 0.008 0.01
Cs

Cs 1 K
= (Cs) + M
r rmax rmax
y= m x +b

Using Microsoft Excel,

The equation of the line is given by:
 y = 3.313305715x + 0.01912464593
1
m= = 3.313305715
rmax
Thus,
1
rmax =
3.313305715
= 0.301813381 mol L • min
KM
b= = 0.01912464593
rmax
Thus,
K M = b × rmax
= (0.01912464593)(0.301813381)
= 5.772074049 × 10 -3 mol L

b) Lineweaver- Burk Plot

Table 2. Lineweaver-Burk Plot Data for the Hydrolysis of Acetylcholme by Dog Serum
1/Cs, L/mol 1/r, L-min/mol
312.5 9.009009
204.0816 6.756757
161.2903 6.993007
125 6.024096
105.2632 5

Linear Form
Figure 4. Lineweaver-Burk Plot, Linear Form
Lineweaver-Burk Plot for the Hydrolysis of
Acetylcholme by Dog Serum

10
9
8
7
6 y = 0.0172x + 3.6342
1/r

5 R² = 0.9146
4
3
2
1
0
0 50 100 150 200 250 300 350
1/Cs

Polynomial Form, n=3

Figure 5. Lineweaver-Burk Plot, Polynomial Form (n=3)
 Lineweaver-Burk Plot for the Hydrolysis of
Acetylcholme by Dog Serum

10

6 y = 3E-06x3 - 0.0017x2 + 0.3308x - 14.674

1/r
R² = 0.9989
4

0
0 50 100 150 200 250 300 350
1/Cs

Polynomial Form, n>3

Figure 6. Lineweaver-Burk Plot, Polynomial Form (n>3)
Lineweaver-Burk Plot for the Hydrolysis of
Acetylcholme by Dog Serum

10

6
1/r

4 y = 1E-08x4 - 7E-06x3 + 0.0011x2 + 0.017x - 1.6946

R² = 1
2

0
0 50 100 150 200 250 300 350
1/Cs

1 KM 1 1
= +
r rmax Cs rmax
y = m x +b
 Using Microsoft Excel,
The equation of the line is given by:

y = 0.01719105838x + 3.63421308
1
b= = 3.63421308
rmax
Thus,
1
rmax =
3.63421308
= 0.2751627321 mol L • min
KM
m= = 0.01719105838
rmax
Thus,
K M = m × rmax
= (0.01719105838)(0.2751627321)
= 4.730338591 × 10 -3 mol
L

c) Eadie-Hofstee Plot
Table 3. Eadie-Hofstee Plot Data for the Hydrolysis of Acetylcholme by Dog Serum
r/Cs, min-1 r, mol/L-min
34.6875 0.111
30.20408 0.148
23.06452 0.143
20.75 0.166
21.05263 0.2

Linear Form
Figure 7. Eadie-Hofstee Plot, Linear Form
Eadie-Hofstee Plot for the Hydrolysis of Acetylcholme
by Dog Serum
0.25

0.2
r, mol/L-min

0.15

0.1 y = -0.0043x + 0.2645

R² = 0.6584
0.05

0
0 5 10 15 20 25 30 35 40
r/Cs, min-1

Polynomial Form, n=3

Figure 8. Eadie-Hofstee Plot, Polynomial Form (n=3)
 Eadie-Hofstee for the Hydrolysis of Acetylcholme by
Dog Serum
0.25

0.2

0.15
r

0.1 y = -0.0002x3 + 0.0144x2 - 0.3987x + 3.8017

R² = 0.7981
0.05

0
0 5 10 15 20 25 30 35 40
r/Cs

Polynomial Form, n>3

Figure 9. Eadie-Hofstee Plot, Polynomial Form (n>3)
Eadie-Hofstee for the Hydrolysis of Acetylcholme by
Dog Serum
0.5

0.4

0.3

0.2
r

0.1
y = -0.0005x4 + 0.0549x3 - 2.2002x2 + 38.646x - 250.89
0 R² = 1
0 5 10 15 20 25 30 35 40
-0.1

-0.2
r/Cs

r
r = -K M + rmax
Cs
y= m x +b

Using Microsoft Excel,

The equation of the line is given by:

y = -4.273141382 × 10 -3 x + 0.2644954792
b = rmax = 0.2644954792 mol L • min

m = -K M = 4.273141382 × 10 -3 mol L

Problem 2.19 The initial rate of reaction for the enzyme cleavage of deoxyguanosine
triphosphate was measured as a function of initial substrate concentration as follows:

Substrate Concentration Initial Reaction Rate

 μMol/L μMol/L
6.7 0.30
3.5 0.25
1.7 0.16

a) Calculate the Michaelis-Menten constants of the above reaction.

Solution:

Using Langmuir Plot:

Table 4: Langmuir Plot Data for the Enzymatic Cleavage of Deoxyguanosine triphosphate
Cs, μmol/L Cs/r,min
6.7 22.33333
3.5 14
1.7 10.625

Linear Form
Figure 10. Langmuir Plot Data, Linear Form
Langmuir Plot for the Enzymatic Cleavage of
Deoxyguanosine triphosphate
25

20
Cs/r, min

15 y = 2.3722x + 6.2429
R² = 0.9937
10

0
0 1 2 3 4 5 6 7 8
Cs, μmol/L

Polynomial Form, n>2

Figure 11. Langmuir Plot Data, Polynomial Form (n>2)
Langmuir Plot for the Enzymatic Cleavage of
Deoxyguanosine triphosphate
25

20

15 y = 2.3722x + 6.2429
Cs/r

R² = 0.9937
10

0
0 1 2 3 4 5 6 7 8
Cs

Same R2 value was generated for polynomial form of order greater than 2 (n>2)

Cs 1 K
= (Cs) + M
r rmax rmax
y= m x +b
 Using Microsoft Excel,
The equation of the line is given by:
y = 2.372227997x + 6.242940055
1
m= = 2.372227997
rmax
Thus,
1
rmax =
2.372227997
μmol
= 0.4215446412 L • min
KM
b= = 6.242940055
rmax
Thus,
K M = b × rmax
= (6.242940055)(0.4215446412)
μmol
= 2.631677926 L
Using Lineweaver-Burk Plot:
Table 5: Lineweaver-Burk Plot Data for the Enzymatic Cleavage of Deoxyguanosine
triphosphate
1/Cs, L/μmol 1/r, L-min/μmol
0.149254 3.333333
0.285714 4
0.588235 6.25

Linear Form
Figure 12. Lineweaver-Burk Plot, Linear Form
Lineweaver-Burk Plot for the Enzymatic Cleavage of
Deoxyguanosine triphosphate
7
6
1/r, L-min/μmol

5
y = 6.7758x + 2.2168
4 R² = 0.9922
3
2
1
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
1/Cs, L/μmol

Polynomial Form, n>2

Figure 13. Lineweaver-Burk Plot, Polynomial Form (n>2)
 Lineweaver-Burk Plot for the Enzymatic Cleavage of
Deoxyguanosine triphosphate
7
6
5
y = 6.7758x + 2.2168
4 R² = 0.9922
1/r

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

Same R2 value was generated for polynomial form of order greater than 2 (n>2)

1 KM 1 1
= +
r rmax Cs rmax
y = m x +b

Using Microsoft Excel,

The equation of the line is given by:
y = 6.775766909x + 2.216782066
1
b= = 2.216782066
rmax
Thus,
1
rmax =
2.216782066
μmol
= 0.4511043352 L • min
KM
m= = 6.775766909
rmax
Thus,
K M = m × rmax
= (6.775766909)(0.4511043352)
μmol
= 3.056577827 L

Using Eadie-Hofstee Plot:

Table 6: Eadie-Hofstee Plot Data for the Enzymatic Cleavage of Deoxyguanosine triphosphate
r/Cs, min-1 r, μmol/L-min
0.044776 0.3
0.071429 0.25
0.094118 0.16

Linear Form
Figure 14. Eadie-Hofstee Plot, Linear Form
Eadie-Hofstee Plot for the Enzymatic Cleavage of
Deoxyguanosine triphosphate
0.35
0.3
r, μmol/L-min

0.25
0.2
0.15 y = -2.8096x + 0.4336
R² = 0.9566
0.1
0.05
0
0 0.02 0.04 0.06 0.08 0.1
r/Cs, min-1

Polynomial Form, n>2

Figure 15. Eadie-Hofstee Plot, Polynomial Form (n>2)
Eadie-Hofstee Plot for the Enzymatic Cleavage of
Deoxyguanosine triphosphate
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

Same R2 value was generated for polynomial form of order greater than 2 (n>2)
 r
r = -K M + rmax
Cs
y= m x +b

Using Microsoft Excel,

The equation of the line is given by:
y = -2.809617888x + 0.433641801
μmol
b = rmax = 0.433641801 L • min
μmol
m = -K M = 2.809617888 L

Based from the R2 values of the three plots, the Langmuir Plot showed the highest R2 value.
Thus,

μmol
K M = 2.631677926 L
μmol
rmax = 0.4215446412 L • min