Enzyme Kinetics

PROBLEM SOVING
Sample Problems:
According to the Michaeli’s-Menten eq’n.
𝑉
What is the ratio when S = 4 Km
𝑉𝑚𝑎𝑥

Given:
S = 4 Km
𝑉
Required:
𝑉𝑚𝑎𝑥
𝑉 [𝑆]
Solution: =
𝑉𝑚 𝐾𝑚 +[𝑆]
𝑉 4𝐾𝑚
=
𝑉𝑚 𝐾𝑚 +4𝐾𝑚
𝑉 4𝐾 4
= 𝑚 = 𝑜𝑟 𝟎. 𝟖
𝑉𝑚 5𝐾𝑚 5
An enzyme and substrate with equal initial concentration of 5 𝜇M are allowed to interact.
After some time the following velocities have been reported depending on substrate
concentration:
[S], mM V, mM/s
0.02 10.83
0.04 18.57
0.07 26.76
0.1 32.5
0.15 39
0.2 43.33
0.3 48.75
0.5 54.17
0.7 56.88

a) Draw the Michaelis – Menten, Eadie – Hofstee, and Hanes – Woolf plot for the data above.
b) Get Km and Vmax for each plot
 Solution:
(For Hanes – Woolf) (For Eadie – Hofstee) (For Michaelis – Menten)
[𝑆] 𝑉 [S] & V
[S] & &V
𝑉 [𝑆]

1
Slope: 59.893
Slope: 0.015071848 = Slope: -0.1000043327 = Vm
𝑉𝑚 Y-int.: 22.913
𝐾𝑚 Y-int.: 65.0087114 = 𝐾𝑚
Y-int.: 1.555329211x10-3 =
𝑉𝑚
Vm = 58 mM/s
Vm = 0.1000043327 mM/s
Vm = 66.31863319 mM/s Km = 0.1 mM
Km = 65.0087114 mM
Km = 0.1013147374 mM
Plot:
The following kinetic data were obtained for an enzyme in the absence of
inhibitor (1), and in the presence of two different inhibitors (2) and (3) at 5mM
concentration. Assume total enzyme concentration is the same in each
experiment.
a) Determine Vmax and Km for the enzyme.
b) Determine the type of inhibition and the K1 for each inhibitor.
[S] (1) (2) (3)
(mM) v (µmol / mL-sec) v (µmol / mL-sec) v (µmol / mL-sec)
1 12 4.3 5.5
2 20 8 9
4 29 14 13
5 35 21 16
12 40 26 18
Given:
[S] (1) (2) (3)
(µM) v (µmol / mL-sec) v (µmol / mL-sec) v (µmol / mL-sec)
1x103 12 4.3 5.5
2x103 20 8 9
4x103 29 14 13
5x103 35 21 16
12x103 40 26 18

Required:
a) Km and Vm
b) Type of inhibition and K1 for each inhibitor
Solution:
𝟏 (1) (2) (3)
𝟏 𝟏 𝟏
[𝑺]
𝑽 𝑽 𝑽
1x10-3 0.0833 0.23255 0.181818
5x10-4 0.05 0.125 0.1111
2.5x10-4 0.03448 0.071428 0.07692
2x10-4 0.02857 0.047619 0.0625
8.3x10-5 0.025 0.03846 0.05556
a) For I1 For I2
Slope: 65.14633298 Slope: 218.1063913
y-int.: 0.01778150101 y-int.: 0.01432934131

1 1
y-int. = y-int. =
𝑉𝑚 𝑉𝑚
1 1
0.01778150101 = 0.01432934131 =
𝑉𝑚 𝑉𝑚
µmol µmol
Vm = 56.23822193 Vm = 69.78687843
𝒎𝑳−𝒔𝒆𝒄 𝒎𝑳−𝒔𝒆𝒄
𝐾𝑚 𝐾𝑚
slope = slope =
𝑉𝑚 𝑉𝑚
𝐾𝑚 𝐾𝑚
65.14633298 = 218.1063913 =
56.23822193 69.78687843
Km = 3663.713932 Km = 15220.96421
b) For I1 & I3
NON – COMPETITIVE

For I1 & I2
1𝑥103 𝜇𝑀
COMPETITIVE I1 = I2 = 5mM x = 5000µM
1𝑚𝑀

𝑉𝑚 56.23822193
V2 = ≈ 4.3 =
[𝐼]
1+ 𝐾
𝐾𝑚
1+ 𝑆 1+
[5000]
1+
3663.713932
𝐼 KI 1𝑥103

KI = 2771.090846 µM
 𝑉𝑚 [𝑆]
V3 = [𝐼]
𝐾𝑚 1+ 𝐾 +[𝑆]
𝐼

56.23822193 1𝑥103
5.5 =
(3663.713932) 1+ [5000]
𝐾
+ 1𝑥103
𝐼

KI = 3293.867117 𝝁M