Exercise 5.9 - Addition of Enzyme Inhibitors

LEVEL II

Materials

Procedure

  1. Tyrosinase is inhibited by compounds that complex with copper, as well as by benzoic acid and cyanide. To determine the inhibitory effects of benzoic acid and cyanide, set up a series of tubes as indicated.

  2. Using one tube at a time, add 0.5 ml of the enzyme dilution previously calculated to yield 10 micromoles of dopachrome in 2-3 minutes. For each tube, measure the time required to convert 10 micromoles of DOPA to dopachrome. Enter those times in the table below. Compute the reaction velocity for each substrate concentration:

    Tube #
    Time (Minutes)
    Final [DOPA] nM
    Velocity
    Micromoles/Minute
    1 Benzoic Acid

    6.67

    2 Inhibited

    6.00

    3 Series

    5.33

    4

    4.67

    5

    4.00

    6

    3.33

    7

    2.67

    8

    2.00

    9

    1.33

    10

    0.67

    11

    0


    12 KCN

    6.00

    13 Inhibited

    5.33

    14 SERIES

    4.67

    15

    4.00

    16

    3.33

    17

    2.67

    18

    2.00

    19

    1.33

    20

    0.67

    21

    0

  3. Calculate the values for 1/s and 1/v for each of the corresponding s and v in the table below. Plot 1/v vs 1/s for the presence of benzoic acid and a second plot for the presence of KCN. Compute the values of V and K_m for the presence of each inhibitor. Determine whether these inhibitors are competitive, non-competitive or uncompetitive.

Benzoic Acid Inhibition
Tube #
8 mM DOPA
8 mM Benzoic Acid
Buffer
1
2.0
0.5
0
2
1.8
0.5
0.2
3
1.6
0.5
0.4
4
1.4
0.5
0.6
5
1.2
0.5
0.8
6
1.0
0.5
1.0
7
0.8
0.5
1.2
8
0.6
0.5
1.4
9
0.4
0.5
1.6
10
0.2
0.5
1.8
11
0
0.5
2.0
KCN Inhibition
Tube #
8 mM DOPA
8 mM KCN
Buffer
12
1.8
0.5
0.2
13
1.6
0.5
0.4
14
1.4
0.5
0.6
15
1.2
0.5
0.8
16
1.0
0.5
1.0
17
0.8
0.5
1.2
18
0.6
0.5
1.4
19
0.4
0.5
1.6
20
0.2
0.5
1.8
21
0
0.5
2.0

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu