2.15. Eadie 2.15. Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by dog serum (source of enzyme) in the absence and presence of prostigmine (inhibitor), 1.5 x 10-7 mol/L and obtained the following data: Substrate
Initial Reaction Rate
Rate (mol/L-min)
Concentration, Cs
Absence of
Presence of
(mol/L)
Prostigmine
Prostigmine
0.0032
0.111
0.0049
Cs/r
Cs/r
(Uninhibited)
(Inhibited)
0.059
0.028828829
0.05423729
0.148
0.071
0.033108108
0.06901408
0.0062
0.143
0.091
0.043356643
0.06813187
0.008
0.166
0.111
0.048192771
0.07207207
0.0095
0.2
0.125
0.0475
0.076
a. Is prostigmine competitive or noncompetitive inhibitor? b. Evaluate the Michaelis-Menten kinetic parameters in the presence of inhibitor by employing the Langmuir Plot. Answers:
Langmuir Plot of Hydroly Hydrolysis sis of Acetylcholine 0.09 0.08
y = 2.9883x + 0.0489
0.07 0.06 y = 3.3133x + 0.0191
0.05 0.04
Cs/r
0.03 0.02
-K MI MI
-K M
0.01 0 -0.02
-0.015
-0.01
-0.005
-0.01 Cs
Without Inhibitor
0
0.005
0 .0 1
0.015
With Inhibitor
a. Based a. Based from the graph, the values of Km with and without the presence of inhibitor is not equal. Therefore, prostigmine is a competitive inhibitor. inhibitor . 1|Biochemical Engineering
10/02/2017
Orpilla, Jerryson M.
BSChE-5
b. Langmuir Equation (With inhibitor): Cs r
y
1
Cs
r max
2.9883x
1
K m r max
0.0489
2.9883
r
max
rmax
mol
0.3346 L
K mi
min
0.0489
r max
K mi
0.01636
mol L
2.19. The 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., J.BioI.Chern., 233, 159, 1958): Substrate Concentration
Initial Reaction Rate (µmol/L-
(µmol/L)
min)
6.7
0.3
0.149254 3.333333
3.5
0.25
0.285714
4
1.7
0.16
0.588235
6.25
1/Cs
1/r
a. Calculate the Michaelis-Menten constants of the above reaction. b. When the inhibitor was added, the initial reaction rate was decreased as follows: Substrate Concentration
Initial Reaction Inhibitor
Rate (µmol/L-
(µmol/L)
min)
6.7
146
0.11
9.090909
3.5
146
0.08
12.5
1.7
146
0.06
16.66667
(µmol/L)
1/r
Is this competitive inhibition or noncompetitive inhibition? Justify your answer by showing the effect of the inhibitor graphically. [Contributed by Professor Gary F. Bennett, The University of Toledo, Toledo, OH] 2|Biochemical Engineering
10/02/2017
Orpilla, Jerryson M.
BSChE-5
Answers:
Lineweaver-Burk Plot of Enzymatic Cleave Lineweaver-Burk of Deoxyguanosine Triphosphate y = 6.7758x + 2.2168
7 6 5 r / 1
4 3 2 1 0 0
0.1
0.2
0 .3
0 .4
0 .5
0 .6
0 .7
1/Cs
a. Lineweaver-Burk Equation 1 r
y
K m 1
r max C s
1
r max
6.7758 x 2.2168
1
2.2168
r
max
rmax
mol
0.451 L
K m
min
6.7758
r max
K m
3|Biochemical Engineering
3.0566
mol L
10/02/2017
Orpilla, Jerryson M.
BSChE-5
b. Based from the graph, the values of Km with and without inhibitor are almost the same. Moreover, the y-intercepts, rmax of the Lineweaver-Burk for both inhibited and uninhibited are not equal. Therefore, it is a noncompetitive inhibition.
Lineweaver-Burk Plot of Enzymatic Cleavage of Lineweaver-Burk Deoxyguanosine Triphosphate 18 y = 16.68x + 7.0637
