Exercise 5.9 - Addition of Enzyme Inhibitors

LEVEL II

Materials

• Enzyme Extract
• 8 mM L-DOPA
• 8 mM Benzoic Acid
• 8 mM KCN
• 0.1 M Citrate Buffer, pH 6.6

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 for the presence of each inhibitor. Determine whether these inhibitors are competitive, non-competitive or uncompetitive.

Return to Table of Contents