Laboratory Notes for BIO 1003

© 30 August 1999, John H. Wahlert & Mary Jean Holland


ENZYMES

Proteins That Act As Catalysts

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Cells do not have the options of heat and vigorous stirring to speed up chemical reactions by making atoms and molecules bump into each other more often and with more force. Instead they make protein catalysts called enzymes that provide surfaces on which reactants encounter one another in the proper orientation, and the enzymes may even weaken bonds that are to be broken. Enzymes have high specificity--only specific reactants will fit on their surfaces and balance the local surface charges of the place called the active site. The particular set of reactants for an enzyme are called its substrate(s). Enzymes, like all catalysts, are not changed by the reaction they catalyze; thus, when all the reactants have been converted to products, the original enzyme molecules remain.

Enzyme function depends on the environment in which the reaction is occurring. Such factors as temperature and pH can influence greatly the ability of an enzyme to do its job. The following series of experiments illustrates the principles of enzymatically catalyzed reactions in biological systems. We will be using the enzyme catecholase (or catechol oxidase) that is common in plants; it is present in the freshly squeezed potato juice that has been provided. The substrates of catecholase are catechol and oxygen. The substrates react with one another within the active site of the enzyme. The products formed by this reaction are benzoquinone and water; since benzoquinone has a brown color, you can see that the reaction has taken place. This is called the fruit browning reaction. Benzoquinone inhibits the growth of microorganisms and prevents damaged fruit from rotting. In undamaged cells catecholase is stored in vesicles and does not interact with catechol.

In the presence of the enzyme catacholase:

catechol  +  ½O2  arrow  benzoquinone  +  H2O

In all of the experiments below, mix the ingredients as specified, and stir the mixture with a vortex genie or by tapping the side of the tube near the bottom; incubate mixtures at 40 degrees C, unless otherwise specified.

If you doubted the purity of the enzyme, how could you find out if there is anything else in the potato extract besides catecholase?

 

I. Formation and Detection of Benzoquinone

This experiment establishes the standards for appearance when the reaction has or has not taken place. Use distilled water where H2O is indicated.

Mix Initial color Color change (after 5 min)
10 drops potato extract with
10 drops 1% catechol (experiment)
   
10 drops potato extract with
10 drops H2O (control)
   
10 drops 1 % catechol with
10 drops H2O (control)
   

How would you define the concepts of a control and of an experiment?

 

II. Enzyme Specificity

Hydroquinone is structurally similar to but not the same as catechol. In this experiment we will see if the enzyme can also convert it to benzoquinone, or if the enzyme is simply too specific in its design to interact with the similar but not identical molecule.

Mix

Initial color Color change (after 5 min)
10 drops potato extract with
10 drops 1% catechol
   
10 drops potato extract with
10 drops hydroquinone
   
10 drops hydroquinone with
10 drops H2O (control)
   

 

III. Effect of Temperature on Enzyme Activity

Enzymes are designed to function within a specific temperature range. You might make some guesses as to the outcome for the following temperatures below. Keep in mind that the structures that produce the enzyme are fruit and potato; on the Celsius temperature scale 0 degrees is freezing, 22 degrees is room temperature, and 100 degrees is boiling. The hotter it is, the more rapidly molecules are vibrating and moving about. Collisions of water molecules with the enzyme could undo its folded structure that is held in position by hydrogen bonds; when this happens the molecule is said to have been denatured. What would happen to the active site in this case? At low temperatures molecules move very slowly, and the chance of their colliding and reacting with one another is very low. Note that the potato extract has been kept on ice. Why?

Write down your hypothesis about the effect of temperature on enzyme activity:

 

Set up 6 pairs of test tubes with 10 drops of potato extract in one set and 10 drops of 1% catechol in the other. Incubate them for 5 minutes, one pair in each of the 5 temperature environments indicated below. Then mix the potato extract and catechol together; record the initial color, and put that tube back in the controlled temperature environments. Record the color again after 5 minutes.

Temperature

Initial color

Color change
(5 min. after adding catechol)

0 degrees C

   

22 degrees C

   

40 degrees C

   

60 degrees C

   

80 degrees C

   

100 degrees C

   

Do the results support your initial hypothesis?

 

Which temperatures denature the enzyme so that it cannot recover.

 

What is the white precipitate that forms at 100 degrees C?

 

IV. Effect of pH on Enzyme Activity

The pH scale measures the numbers of hydrogen ions (each with a +1 charge) present in solution. The scale in biological systems runs from 0 (strongly acidic) to 14 (strongly basic). 7, the neutral point, is the pH of pure water. Recall that the folded structure of protein molecules is in large part due to hydrogen bonding between oppositely charged parts of the polypeptide chain. Other ions in solution could replace the matching parts of the chain, and make it come undone. When this happens, what becomes of the active site?

Write down your hypothesis about the effect of the range of pH on enzyme activity:

 

Set up 7 test tubes with 40 drops of a different buffered pH solution in each. Add 10 drops of potato extract to each and stir. Then add 10 drops of 1% catechol to each and stir.

pH

Initial color

Color change (5 min after adding catechol)

pH 2

   

pH 4

   

pH 6

   

pH 7

   

pH 8

   

pH 10

   

pH 12

   

Do the results support your hypothesis?

 

At What pH or range of pH values does the enzyme work best?

 

Why does putting lemon juice on freshly sliced fruit retard its browning?

 

 

V. Necessity of Cofactors for Enzyme Activity [Optional]

Many enzymes work only if there is a cofactor associated with them. Catecholase requires copper as a cofactor. PTU is a chemical that has a higher affinity for copper than does catecholase, and it takes the copper away from the enzyme. You can observe what happens to the function of the enzyme when its cofactor is removed. We refer to cofactors that our bodies need as vitamins and minerals.

Mix

Initial color

Color change (5 min after adding catechol)

Tube A
10 drops potato extract
5 crystals PTU and mix.
After 5 minutes add
10 drops 1% catechol.

   

Tube B
10 drops potato extract.
After 5 minutes add
10 drops 1% catechol.

   

Which tube, A or B, is the control in this experiment?

 

What hypothesis are you testing in the experiment described above.

 


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Last updated 30 August 1999 (JHW)