Biology 1130 - the Effect of Competitive Inhibitors on Catechol Oxidase

The Effect of Competitive Inhibitors on Catechol Oxidase

Alejandra Ramos

Biology 1130

Section: 283

Abstract

The Effects of competitive inhibitors on Catechol Oxidase. Nicholas Siepert, 2015, Functional Biology, Texas State University, San Marcos, TX 78666. 

        Catechol Oxidase is an enzyme responsible for the defense mechanism plants, fruits, and vegetables have to change in pigment, in this case melanin, when bruised, cut, or exposed to other environmental factors. In this experiment, a competitive inhibitor-chemicals that delay or eliminate a reaction-is introduced, parahydroxybenzoic acid is used in this experiment. To measure the reaction speed of both chemical and substrate together, four different amounts of P.H.B. acid were mixed with enzyme solution of catechol oxidase taken from potato homogenate for pigment absorption observation. It was gathered from the experiment result, after a series of three trials per tube, for a total of 12 trials that the P.H.B. acid when mixed with an enzymatic solution decreased the rate of change in the color of the solution, increasing still, but much slower.

Introduction

An enzyme, which is also known as a catalyst, is assigned within a living cell to preform a multitude of different chemical reactions at a faster rate, but they do so without being changed or altered in anyway during the process. Enzymes can do so by lowering the activation energies, where as the remainder of the chemical processes act according to whatever environmental parameters, if any that are present. All enzymes also contain: a set of activators, chemicals that bind for the enzyme to become active, a set of cofactors, which are a non-protein substance bound to the active sites of enzymes prior to the beginning of a reaction that are often metal ions, and coenzymes, which is an organic form of a cofactor.

Enzymes, after binding with a substrate, are joined to increase the odds of a reaction-taking place. When a catalyst’s active sites bind with a substrate, it will be that substrate that is altered or changed to form a new product, and not the catalyst.

Catechol Oxidase is one of three copper-containing enzymes that are typically found in the living cells of plants, fruits, and vegetables. Catechol Oxidase, or catalase, increases the reactions that form Quinones, a natural occurrence a plant operates on as a means of defense against exposure to insect and, or insecticides, and other environmental manifestations.

Whenever Catechol Oxidase is exposed to oxygen, the enzyme begins to break down, by stripping the catechol of it’s electrons and hydrogen ions to form benzoquinone, a pigment, in this experiment involving competitive inhibitors, melanin. This is the same mechanism of catechol oxidase in motion that turns bruised or cut fruits and vegetables to turn brown. This experiment utilized the catechol oxidase of potato homogenate (Mosby’s Medical Dictionary, 2007), also known as, Tyrosinase (Richter, 1934). Inhibitors, or in this case, competitive inhibitors, are chemicals that delay or entirely prevent a certain reaction from taking place. Certain studies are currently being tested and utilized in medicine by introducing inhibitors into human tissues to stunt the growth of cancerous cells in the human body (Xiaojie et al., 2014).

The goal of this lab is to introduce the coenzyme’s activity to a competitive inhibitor, in this experiment the poisonous chemical, parahydroxybenzoic acid, and observe how the reaction was affected through the changes in pigmentation. The most important questions that arise from this experiment are: was the data readily observable, meaning how fast did are we able to see a change, if any? Does the amount of substrate or the dilutions of chemicals determine the speed of the reaction process? Lastly, will our data support our hypotheses? Accordingly, our null hypothesis for this experiment states, “The enzyme solution will not be effected by the competitive inhibitor.” Whereas our alternative hypothesis states, “The enzyme solution will be effected by the competitive inhibitor.”

Materials and Methods

Since poisonous chemicals are involved, gloves and goggles, as well as the appropriate attire are mandatory for the duration of the experiment. It is important that no cuvette should have more than 4mL of liquid and all should be covered with Parafilm after mixing to prevent further exposure to poisonous chemicals. All fume hoods should remain closed after handling and while disposing of chemicals. A series of three trials will be conducted for every tube in the experiment, for a total of 14 tubes. 12 of the 14 tubes will contain enzyme catechol, substrate catechol oxidase and competitive inhibitor parahydroxybenzoic acid, and the remaining two will contain only water and catechol oxidase, which will serve as the starting points and “blanks” between the trials.