Catechol Oxidase Lab Report

In the lab, Inhibiting the Action of Catechol Oxidase we had to investigate what type of enzyme inhibition occurs when an inhibitor is added. Catechol oxidase is an enzyme in plants that creates benzoquinone.Benzoquinone is a substance that is toxic to bacteria. It is brown and is the reason fruit turns brown. Now, there are two types of inhibitors, the competitive inhibitor and non-competitive inhibitor. For an enzyme reaction to occur a substrate has to bind or fit into the active site of the enzyme. In competitive inhibition there is a substrate and an inhibitor present, both compete to bind to the active site. If the competitive inhibitor binds to the active site it stops the reaction. A noncompetitive inhibitor binds to another region

of the enzyme and alters the shape of the active site which prohibits the substrate from binding to the active site. In this lab we utilized the inhibitor phenylthiourea (PTU) which is associated with copper, a cofactor in making catechol oxidase work. We were testing how phenylthiourea would affect the binding of copper and catechol. We hypothesize that when phenylthiourea combines with copper it will competitively inhibit catechol. We had to obtain data which allowed us to test our hypothesis of how phenylthiourea affects the binding of copper and catechol. In this lab we will be adding distilled water, potato extract, and catechol to three test tubes. We will also be adding the inhibitor phenylthiourea to two of the test tubes and mixing each tube when all the contents are added. If phenylthiourea competitively inhibits catechol, then adding more catechol will cause a reaction to occur.

Materials and Methods
In order to test whether phenylthiourea would affect the binding of copper and catechol we obtained three test tubes, distilled water, potato extract, phenylthiourea and catechol. In sequential order we added 5 mL of distilled water, 0.5 mL of potato extract, 0.5 mL of phenylthiourea, 0.5 mL of distilled water, and 0.5 mL of catechol to test tube number one. Note that we added the substrate catechol to the test tubes last because we did not want the reaction to occur while we were still adding the other solutions to the test tubes. We mixed test tube number one and proceeded to add the solutions to the following test tube. We added 5 mL of distilled water, 0.5 mL of potato extract, 0.5 mL of phenylthiourea, and 1 mL of catechol to test tube number two. The concentration of catechol was increased in test tube 2 because we were testing if adding more catechol would outcompete the competitive inhibitor phenylthiourea. After mixing test tube number two we moved onto test tube number three. We added 5 mL of distilled water, 5 mL of 0.5 potato extract, 1 mL of distilled water, and 0.5 mL of catechol to test tube number three. After we mixed test tube number three, we recorded our observations on a data table. The control in the experiment was test tube number three because we did not add the inhibitor phenylthiourea.