An AP Biology Lab: Inhibiting the Action of Catechol Oxidase

Lab:

Inhibiting the Action of Catechol Oxidase

By: Kimberly G.

Introduction:

In this lab, Mr. Greene 's sixth period AP Biology class split into groups "to investigate inhibition of enzyme activity by specific chemicals called inhibitors" (1). Group three pondered this lab 's inhibitor, phenylthiourea (PTU).

Is it a competitive inhibitor? That is an inhibitor that literally "competes" with the substrate by mimicking it, and thus "wins" the position at the active site of the enzyme. The blocked substrate in turn cannot join with the active site, causing the enzyme 's activity to be hindered. This inhibition can be reversed, but only if the substrate 's concentration levels are increasing raised while the inhibitor 's concentration levels are remained stable.

Or is PTU a noncompetitive inhibitor? A noncompetitive inhibitor also joins with the enzyme, but it joins to a site other than the active site. This action changes the enzyme 's nature causing the enzyme to lose its catalytic properties. During this inhibition, the substrate cannot reverse the inhibitor 's actions, so increasing the concentration of the substrate is fruitless.

PTU, an inhibitor of catechol oxidase, must join with copper, as a cofactor, in order for the catechol oxiadase to be active, and PTU is known for doing just that. Group Three hypothesized that PTU is in fact a noncompetitive inhibitor based on their knowledge of the nature of competitive and noncompetitive inhibitors.

The Group then went on to predict that there won 't be a reaction when the substrate 's concentration is raised because the Group predicted that it is a noncompetitive inhibitor.

Materials and Methods:

? Test tube rack

? 3 small test tubes

? Calibrated 5-ml pipette

? Distilled water

? Potato extract

? Catechol

? Disposable Pasteur pipettes

? Pipette bulb

? Phenylthiourea (PTU)

? Optional disposable gloves

Using Table 3.1 prepare three test tubes. Add the solutions: water, potato extract, PTU, in the order the table suggests, from left to right.

Table 3.1

Contents of the Three Experimental Tubes

TubeDistilled WaterPotato ExtractPTUDistilled WaterCatechol

15 ml10 drops10 drops10 drops10 drops

25 ml10 drops10 drops------------------20 drops

35 ml10 drops------------------20 drops10 drops

Results:

Group Three 's results are depicted by the following table, Table 3.2. The first two tubes did not react at all, and the third tube turned brown.

Table 3.2

Results of Inhibition Experiment

TubeReaction

1------------------

2------------------

3Turned Brown

Discussion:

In this lab, test tubes 1 and 3 were the constant tubes. Tube three was not, and therefore the catechol 's concentration was increased to confirm or refute Group Three 's hypothesis. The substrate, added last, was done so because the substrate would have initiated the enzyme 's reaction instantly if the inhibitor was not previously there to hinder its reaction, which in this case, it was present. And, considering the groups needed to see whether or not the inhibitor was competitive or noncompetitive, that wouldn 't have been beneficial.

So, is PTU a competitive inhibitor or a noncompetitive inhibitor? Group three hypothesized that it is a noncompetitive inhibitor, and went on to predict that there would no reaction after an increase in the substrate 's concentration. Using the data from Table 3. 1, Table 3.2, and knowledge of competitive and noncompetitive inhibitors, Group Three deduced that PTU is, in fact, a noncompetitive inhibitor.

So, Group Three 's hypothesis was supported, and since there was no reaction after the substrate 's concentration was raised, the prediction was confirmed, too.

There are no errors that could have affected Group Three 's results to be documented besides general lab procedure errors.

References:

1. Giles Morgan, J. and Brown Carter, M.E. Investigating Biology 2nd ed. Menlo Park, CA: Benjanin/Cummings, 1996.

References: 1. Giles Morgan, J. and Brown Carter, M.E. Investigating Biology 2nd ed. Menlo Park, CA: Benjanin/Cummings, 1996.