Escolar Documentos
Profissional Documentos
Cultura Documentos
In an attempt to control the zebra mussel, several compounds have been tried as potential inhibitors of a key
enzyme in NTP growth. Below are the v vs. [S] data for these inhibitors. Based on these data, determine (i)
KI for each of inhibitors (UNITS PLEASE!) and (ii) the type of inhibitor each of them is.
[S]
0.2
0.25
0.33
0.5
1
2
2.5
3.33
4
5
No Inh
16.7
20
25
33.3
50
66.7
71.4
76.9
80
83.3
[Inh A] 6 uM
6.25
7.7
10
14.3
25
40
45.5
52.6
57.1
62.5
[Inh B] 4 mM
10
11.1
12.5
14.3
16.7
18.2
18.5
18.9
19
19.2
In order to answer these questions, you must first plot the data in a reciprocal plot and then calculate slope
(Km/Vm), and y- intercept (1/Vmax) and x-intercept (1/Kmapparent):
No Inh
0.06
0.05
0.04
0.03
0.02
0.01
0.01
0.01
0.01
0.01
[Inh A] 6 uM [Inh B] 4 mM
0.16
0.10
0.13
0.09
0.10
0.08
0.07
0.07
0.04
0.06
0.03
0.05
0.02
0.05
0.02
0.05
0.02
0.05
0.02
0.05
0.18
0.16
No Inh
0.14
[Inh A] 6 uM
0.12
[Inh B] 4 mM
0.10
1/v
1/[S]
5.00
4.00
3.03
2.00
1.00
0.50
0.40
0.30
0.25
0.20
0.08
0.06
0.04
0.02
0.00
0.00
m (Km/Vm)=
b=
x intercept
Vm
Km
0.01
0.01
-1.01
100.00
0.99
Ki
Type
NA
0.03
0.01
-0.34
101.01
2.97
1.00
2.00
3.00
1/[S] (M-1)
4.00
0.01
0.05
-5.03
20.04
0.20
3uM
1 mM
Competitive Uncompetitive
Since Inhibitor A only affects Slope and apparent Km, you know that it is a competitive inhibitor, whereas
Inhibitor B affects the 1/Vmax and apparent Km, you know that this is an uncompetitive inhibitor.
5.00
6.00
To calculate KI, you must know the Lineweaver-Burk equations for each inhibitor type:
Competitive:
1/v = 1/Vmax + Km/Vmax *(1+[I]/KI)* 1/[S]
Thus, slope = Km/Vmax *(1+[I]/KI)
Therefore:
Slope[I] = Slope[I]=0 * (1+[I]/KI)= Slope[I]=0 + (Slope[I]=0 *[I]/KI)
0.03= .01 + (0.01*6M/KI)
0.02 = .06M/KI
0.02 KI = 0.06 M
KI = 3M
Uncompetitive:
1/v = 1/Vmax *(1+[I]/KI)+ Km/Vmax * 1/[S]
Thus intercept= 1/Vmax *(1+[I]/KI)
Therefore:
Intercept[I]= Intercept[I]=0 * (1+[I]/KI)= Intercept [I]=0 + (Intercept [I]=0 *[I]/KI)
0.05 = .01 + (.01[I]=0 *[4mM]/KI)
0.04 = .04mM/KI
0.04KI=0.04mM
KI = 1 mM
QUESTION 2
The enzyme laccase catalyzes the reduction of oxygen to water.
4 e- + O2 2 H2O
Even though this is a bi-substrate reaction, the velocity of binding oxygen can be ignored and thus the
kinetic mechanism can be dealt with as:
k1 k3
E + S ES E + P
k2
Just like cytochrome oxidase, laccase is inhibited by the addition of azide (N3-). Below is given the kinetic
data for the inhibition of oxygen reduction by N3-. A) Please analyze this data and enumerate Km, Vmax for
the reaction and KI for N3- inhibition. B) What kind of inhibitor is N3-?
In evaluating KI and the type of inhibition (Hint: be sure to determine KI in two ways), please show
the kinetic mechanism and show the velocity equation for the scheme. Be sure to justify your answers using
velocity equations and/or its derivatives. For complete credit, show ALL work and include ALL
plots/replots.
[S] (M)
[N3]=0
1.50E-04
5.26
2.00E-04
6.81
4.50E-04 13.40
9.00E-04 21.85
1.40E-03 28.20
[N3]=0
0.19
0.15
0.07
0.05
0.04
[N3]=0
[N3]=6 uM
[N3]=30 uM
[N3]=60 uM
[N3]=150 uM
1/v
0.3
0.2
0.1
1/[S]
Then, calculate slope (Km/Vm), and y- intercept (1/Vmax) and x-intercept (1/Kmapparent):
Slope
Y-intercept
X-intercept
Looking at these data, we see both slope and intercept effects, suggesting a noncompetitive inhibition.
However, the x-intercept is not the same for all, thus, this is NOT pure noncompetitive, indicating partial
noncompetitive inhibition
This is verified by looking at the slope and intercept replots.
0.075
0.000100
y-intercept
Slope
0.000075
0.000050
0.050
0.025
0.000025
[I] M
[I] M
As you can see, the -X intercept (= -KI) of the slope and y-intercept replots differ. With the KI for the y
intercept (Vmax)= 58M, and that for the slope being 78M. Thus, the reaction mechanism is:
E+S D
+
I
EKslope
EI +S D
ES E+ P
+
I
EKintercept
ESI