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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

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

1/[S]

6666.67

5000.00

2222.22

1111.11

714.29

[N3]=0

0.19

0.15

0.07

0.05

0.04

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

1/[S]

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

Slope

Y-intercept

X-intercept

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

[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

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