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

0.05
f(x) = 3.31x + 0.02
0.04 R² = 0.88
Cs/r, min

0.03

0.02

0.01

0
0 0 0 0.01 0.01 0.01 0.01 0.01 0.01

Cs, mol/L

Polynomial Form, n=3

Figure 2. Langmuir Plot, Polynomial Form (n=3)
 Object 5

Polynomial Form, n>3

Figure 3. Langmuir Plot, Polynomial Form (n>3)

Object 7

Cs 1 K
= ( Cs ) + M
r r max r max
y= m x +b
Using Microsoft Excel,
The equation of the line is given by:
 y=3 . 313305715x+0. 01912464593
1
m= =3 . 313305715
r max
Thus,
1
r max =
3. 313305715
mol
=0. 301813381
L⋅min
KM
b= =0 . 01912464593
r max
Thus,
K M =b×r max
= ( 0. 01912464593 ) ( 0 .301813381 )
mol
=5. 772074049×10 -3
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 f(x) = 0.02x + 3.63
7 R² = 0.91
6
5
1/r

4
3
2
1
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
9
8 f(x) = 0x^3 - 0x^2 + 0.33x - 14.67
7 R² = 1
6
5
1/r
4
3
2
1
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
9
8 f(x) = 0x^4 - 0x^3 + 0x^2 + 0.02x - 1.69
7 R² = 1
6
5
1/r

4
3
2
1
0
50 100 150 200 250 300 350

1/Cs

1 KM 1 1
= +
r r max Cs r max
y= m x +b
 Using Microsoft Excel,
The equation of the line is given by:
y=0 . 01719105838x+3. 63421308
1
b= =3 . 63421308
r max
Thus,
1
r max =
3. 63421308
mol
=0. 2751627321
L⋅min
K
m= M =0 . 01719105838
r max
Thus,
K M =m×r max
= ( 0. 01719105838 ) ( 0 .2751627321 )
mol
=4 .730338591×10−-3
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 f(x) = - 0x + 0.26

R² = 0.66

0.1

0.05

0
20 22 24 26 28 30 32 34 36

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
f(x) = - 0x^3 + 0.01x^2 - 0.4x + 3.8
0.15 R² = 0.8
r

0.1

0.05

0
20 22 24 26 28 30 32 34 36

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

0.25
f(x) = - 0x^4 + 0.05x^3 - 2.2x^2 + 38.65x - 250.89
R² = 1
0.2

0.15
r

0.1

0.05

0
20 22 24 26 28 30 32 34 36

r/Cs

r=-K M (rCs )+r max

y= m x +b
Using Microsoft Excel,
The equation of the line is given by:

y=-4 .273141382×10-3 x+0 . 2644954792

mol
b=r max =0 . 2644954792
L⋅min
mol
m=-K M =4 . 273141382×10 -3
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 f(x) = 2.37x + 6.24

R² = 0.99
15
Cs/r, min

10

0
1 2 3 4 5 6 7

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 f(x) = 0.15x^2 + 1.12x + 8.31

R² = 1
15
Cs/r

10

0
1 2 3 4 5 6 7

Cs

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

Cs 1 K
= ( Cs ) + M
r r max r max
y= m x +b
 Using Microsoft Excel,
The equation of the line is given by:

y=2 . 372227997x+6. 242940055

1
m= =2 . 372227997
r max
Thus,
1
r max =
2. 372227997
μmol
=0. 4215446412
L⋅min
KM
b= =6 . 242940055
r max
Thus,
K M =b×r max
= ( 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 f(x) = 6.78x + 2.22
5 R² = 0.99
1/r, L-min/μmol

4
3
2
1
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 f(x) = 5.81x^2 + 2.36x + 2.85
5 R² = 1

4
1/r
3
2
1
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 r max Cs r max
y= m x +b
Using Microsoft Excel,
The equation of the line is given by:

y=6 . 775766909x+2. 216782066

1
b= =2 . 216782066
r max
Thus,
1
r max =
2. 216782066
μmol
=0. 4511043352
L⋅min
KM
m= =6 . 775766909
r max
Thus,
K M =m×r max
= ( 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
f(x) = - 2.81x + 0.43
0.25 R² = 0.96
r, μmol/L-min

0.2

0.15

0.1

0.05

0
0.04 0.05 0.06 0.07 0.08 0.09 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
f(x) = - 42.37x^2 + 3.05x + 0.25
0.25 R² = 1
0.2
r

0.15

0.1

0.05

0
0.04 0.05 0.06 0.07 0.08 0.09 0.1

r/Cs

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

r
r=-K M
Cs
+r max( )
y= m x + b
 Using Microsoft Excel,
The equation of the line is given by:
y=-2 . 809617888x+0 . 433641801
μmol
b=r max =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
r max =0. 4215446412
L⋅min