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QUESTION 1

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]=6 M [N3]=30 M [N3]=60 M [N3]=150 M


4.96
3.66
2.81
1.76
6.39
4.75
3.65
2.27
12.37
9.44
7.29
4.37
19.76
15.61
12.09
6.97
25.12
20.36
15.82
8.84

To begin, we again replot the data in a reciprocal plot


1/[S]
6666.67
5000.00
2222.22
1111.11
714.29

[N3]=0
0.19
0.15
0.07
0.05
0.04

[N3]=6 uM [N3]=30 uM [N3]=60 uM [N3]=150 uM


0.20
0.27
0.36
0.57
0.16
0.21
0.27
0.44
0.08
0.11
0.14
0.23
0.05
0.06
0.08
0.14
0.04
0.05
0.06
0.11

[N3]=0
[N3]=6 uM
[N3]=30 uM
[N3]=60 uM
[N3]=150 uM

1/v

0.3

0.2

0.1

-500 500 1500 2500 3500 4500 5500 6500 7500

1/[S]

Then, calculate slope (Km/Vm), and y- intercept (1/Vmax) and x-intercept (1/Kmapparent):

Slope
Y-intercept
X-intercept

[N3]=0 [N3]=6 uM [N3]=30 uM [N3]=60 uM [N3]=150 uM


2.596E-05 2.720E-05 3.764E-05 4.907E-05
7.656E-05
1.695E-02 2.042E-02 2.230E-02 2.824E-02
5.857E-02
-653
-751
-592.3
-575.5
-765

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

-100-75 -50 -25 0 25 50 75 100125150175200 -100-75 -50 -25 0

[I] M

25 50 75 100 125 150 175 200

[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