Catechol Oxidase (Tyrosinase) - Enzymes
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Catechol Oxidase - A Study of Inhibition

An enzyme that destroys some toxins and makes melanin sun screen.

Living cells perform a multitude of chemical reactions very rapidly because of the participation of enzymes. Enzymes are biological catalysts, compounds that speed up a chemical reaction without being used up or altered in the reaction. The material with which the catalyst reacts, called the substrate, is modified during the reaction to form a new product (Fig. 1). But because the enzyme itself emerges from the reaction unchanged and ready to bind with another substrate molecule, a small amount of enzyme can alter a relatively enormous amount of substrate.

The active site of an enzyme will bind with the substrate, Forming the enzyme-substrate complex. It is here that catalysis takes place, and when it is complete, the complex dissociates into enzyme and product or products.

Enzymes are, in part or in whole, proteins and are highly specific in function. Because enzymes lower the energy of activation needed for reactions to take place, they accelerate the rate of reactions. They do not, however, determine the direction in which a reaction will go or its final equilibrium.

Enzyme activity is influenced by many factors. Varying environmental conditions, such as pH or temperature, may change the three-dimensional shape of an enzyme and alter its rate of activity. Specific chemicals may also bind to an enzyme and modify its shape. Chemicals rhat must bind for the enzyme to be active are called activators. Cofactors are nonprotein substances that usually bind to the active site on the enzyme and are essential for the enzyme to work. Organic cofactors are called coenzymes, but other cofactors may simply be metal ions. Chemicals that shut off enzyme activity are called inhibitors and their action can be classified as competitive or noncompetitive inhibition.


Figure 1: Enzyme activity. A substrate or substrates bind to the active site of the enzyme forming the enzymesubstrate complex, which then dissociates into enzyme and product(s). The enzyme may catalyze the addition or removal oi'a molecule or a portion of a molecule from the substrate to produce the product (a), or the enzyme may catalyze the splitting ofa substrate into its component subunits (b).

Review Figure 1, illustrating enzyme activity. There are two ways to measure enzyme activity: (1) determine the rate of disappearance of the substrate and (2) determine the rate of appearance of the product.

In this laboratory, you will use both methods to investigate the activity of catechol oxidase. You will use an inhibitor to influence the activity of catechol oxidase and determine if it is a competitive or noncompetitive inhibitor. Additionally, you will investigate the effect of changing environmental conditions on the rate of amylase activity.


Experimental Method and the Action of Catechol Oxidase

Materials

.What does this have to do with Siamese Cats? Other names for catechol oxidase are diphenol oxidase and tyrosinase. Siamese cats and Himalayan rabbits have a heat-labile tyrosinase. Normal tyrosinase converts the amino acid tyrosine into melanine (black pigment). In these two types of animals with this enzyme that denatures at normal body temperatures, the black is formed only on the colder extremities of the body. This can be tested by looking at Siamese cats that have been left outdoors over winter (all black), or raised in very hot climates (all white). Of course you can also put boots on the cats and get cats with white stockings, or tape down their ears and get white ears..

Introduction

This exercise will investigate the result of catechol oxidase activity. In the presence of oxygen, catechol oxidase catalyzes the removal of electrons and hydrogens from catechol, a phenolic compound found in plant cells. Catechol is converted to benzoquinone, a pigment product. The hydrogens combine with oxygen, forming water (Fig. 2). The pigment products are responsible for the darkening of fruits and vegetables, such as apples and potatoes, after exposure to air.

In this exercise you will use an extract of potato tuber to test for the presence of catechol oxidase and to establish the appearance of the products when the reaction takes place.

Figure 2: The oxidation of catechol. In the presence of catechol oxidase, catechol is converted to benzoquinone. Hydrogens removed from catechol combine with oxygen to form water.

Hypothesis

Before making your hypothesis, review the information given above about the activity of catechol oxidase and construct a hypothesis for its presence or absence in potato extract. Remember, the hypothesis must be testable. It is possible for you to propose one or more hypotheses, but all must be testable.

Prediction

Predict the result of the experiment based on your hypothesis. To test for the presence or absence of catechol oxidase in potato extract, your prediction wouId be what you expect to observe as the result of this experiment (if/then) .


Catechol is a poison! Avoid contact with all solutions. Do not pipette any solutions by mouth. Wash hands thoroughly after each experiment. If a spill occurs, notify the instructor. If the instructor is unavailable, wear disposable gloves and use dry paper towels to wipe up the spill. Follow dry towels with towels soaked in soap and water. Dispose of all towels in the trash.


Procedure

  1. Using Table I, prepare the three experimental tubes. Note that all tubes should contain the same total amount of solution.

    Table I: Contents of the Three Experimental Tubes.

  2. Explain the experimental design: What is the purpose of each of the three test tubes! Which is the control tube! Which is the experimental tube!

  3. Observe the reactions in the tubes and record your observations in the Results section below. Explain your conclusions in the Discussion section.

Results

Record your results (Table II).

Discussion

Explain your results in terms of your hypothesis.


Inhibiting the Action of Catechol Oxidase

Materials

Introduction

This exercise will investigate the inhibition of enzyme activity by specific chemicals called inhibitors. The specific inhibitor used will be phenylthiourea (PTU). To be active, catechol oxidase requires copper as a cofactor. PTU is known to combine with the copper in catechol oxidase and inhibit its enzymatic activity.

An inhibitor molecule affects an enzyme in one of two ways. Competitive inhibition takes place when a molecule that is structurally similar to the substrate for a particular reaction competes for a position at the active site on the enzyme. This ties up the enzyme so that it is not available to the substrate. Competitive inhibition can be reversed if the concentration of substrate is raised to sufficiently high levels while the concentration of the inhibitor is held constant (Figure3).

Figure 3: Action of a competitive inhibitor. (a) Substrate normally can bind to the active site of an enzyme. (b) A competitive inhibitor mimics the substrate and competes for the position at the active site on the enzyme. In noncompetitive inhibition, the inhibitor binds to a site that is not the active site. In so doing, it changes the nature of the enzyme so that its catalytic properties are lost. This can happen in two ways. Either the noncompetitive inhibitor itself physically blocks the access to the active site, or it causes a conformational change in the protein, thus inactivating the active site. Because the substrate molecules cannot reverse the binding of a noncompetitive inhibitor, increasing the concentration of substrate will not reverse the inhibition. (See Figure 2.4 on the next page.)

In the following experiment, you will determine if PTU is a competitive or noncompetitive inhibitor.

Figure 4:

Figure 4: Action ofa noncompetitive inhibitor. The noncompetitive inhibitor binds to the enzyme at a location away from the active site, either blocking access to the active site or changing the conformation of the enzyme, rendering it inactive.

Hypothesis

Hypothesize about the nature of inhibition by PTU.

Prediction

Predict the results of the experiment based on your hypothesis (if/then).

Procedure


PTU and catechol are poisonsl Avoid contact with solutions. Do not pipette any solutions by mouth. Wash hands thoroughly after the experiment. If a spill occurs, notify the instructor. If the instructor is unavailable, wear disposable gloves and use dry paper towels to wipe up the spill. Follow dry towels with towels soaked in soap and water. Dispose of all towels in the trash.


  1. Using Table III, prepare three experimental tubes. Be sure to add solutions in the sequence given in the table (water first, potato extract next, PTU next, etc.).

    Table III:
    Contents of the Three Experimental Tubes:

  2. Which test tube is the control!

  3. Why was the concentration of catechol increased in test tube 2!

  4. Why should the catechol be added to the test tubes last!

  5. Record your observations in the Results section and explain your results in the Discussion section.

Results

Record your results (Table IV).

Discussion:

Explain your results in terms of your hypothesis.


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