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Andrada, Filmar T.
Dulliyao, Van Vesper J.
Cauilan, Kaicee
Lapuebla, Jeremy C.
Necesito, Miko Paul B.
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 (mol/L) Initial Reaction Rate (mol/L)
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) Employing the Langmuir plot
x (Cs) y (r)
Langmuir Plot
0.0032 0.028829
0.0049 0.06
0.033108
0.0062 0.043357 y = 3.3133x + 0.0191
0.05
R² = 0.8837
0.008 0.048193
0.0095 0.0475 0.04
Cs/r
0.03
1
rmax 0.02
slope
0.01
K m y intercept rmax
0
0 0.002 0.004 0.006 0.008 0.01
rmax 0.301814
Cs
Km 0.005765
1
b) Lineweaver-Burk Plot
x (1/Cs) y (1/r)
Lineweaver-Burk Plot
312.5 9.009009
10
204.0816 6.756757
9 y = 0.0172x + 3.6342 R² = 0.9146
161.2903 6.993007 8
125 6.024096 7
105.2632 5 6
1/r
5
4
1
rmax 3
y intercept
2
K m slope rmax
1
0
0 50 100 150 200 250 300 350
rmax 0.275164
1/Cs
Km 0.004733
c) Eadie-Hofstee Plot
x (r/Cs) y (r)
Eadie-Hofstee Plot
34.6875 0.111
0.25
30.20408 0.148
23.06452 0.143
0.2
20.75 0.166
21.05263 0.2 0.15
y = -0.0043x + 0.2645
r
R² = 0.6584
rmax y intercept 0.1
K m slope 0.05
rmax 0.2645 0
Km 0.0043 0 5 10 15 20 25 30 35 40
r/Cs
2
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 (μmol/L) Initial Reaction Rate (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:
In finding the Michaelis-Menten constants, we first determine the highest coefficient of R
by plotting the three different graphs namely Langmuir, Lineweaver-Burk, and Eadie-Hofstee plot.
Cs r
6.7 0.3
3.5 0.25
1.7 0.16
1. Langmuir Plot
Cs Cs/r
Langmuir Plot
6.7 22.33333
30
3.5 14
y = 2.3722x + 6.2429
1.7 10.625 20
Cs/r
R² = 0.9937
10
R2=0.9937 0
0 2 4 6 8
Cs
2. Lineweaver-Burk Plot
Lineweaver-Burk Plot
1/Cs 1/r 8
0.149254 3.333333 y = 6.7758x + 2.2168
6
0.285714 4 R² = 0.9922
1/r
0.588235 6.25 4
R2=0.9922 0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
1/Cs
3
3. Eadie-Hofstee Plot
r/Cs r
Eadie-Hofstee Plot
0.044776 0.3
0.071429 0.25 0.4
r
R² = 0.9566
R2=0.9566 0.1
0
0 0.02 0.04 0.06 0.08 0.1
r/Cs
Among the above graphs, the Langmuir plot displayed the best fit of our data. Using the
Langmuir plot to solve the problem, the equation below shall be used:
1
rmax
slope
K m y intercept rmax
rmax 0.42155
Km 2.631692