LAB TOPIC 6 Enzymes : Catalysts of Life Adapted from Mader, Laboratory Manual:  Biology (9th edition) and  Perry, Morton and Perry, Laboratory Manual for General Biology (8th Edition) Laboratory Objectives:  After completing this exercise, you will be able to Define enzyme, activation energy, enzyme-substrate complex, substrate, product, active site, denaturation

LAB TOPIC 6

Enzymes : Catalysts of Life

Adapted from Mader, Laboratory Manual:  Biology (9^th edition) and 

Perry, Morton and Perry, Laboratory Manual for General Biology (8^th Edition)

Laboratory Objectives: 

After completing this exercise, you will be able to

1. Define enzyme, activation energy, enzyme-substrate complex, substrate, product, active site, denaturation.
2. Discuss the function of an enzyme. 
3. Explain the role of the active site in enzyme specificity and activity.
4. Identify the substrate, enzyme, and product in the reaction studied today.
5. Predict the effect of temperature on the rate of chemical reactions in general and on         enzymatically controlled reactions in particular. 
6. Predict the effect of enzyme and substrate concentration on an enzymatic reaction.  
7. Predict the effect that an atypical pH may have on an enzymatic reaction. 

Before you begin the activities in this Lab Topic, at your instructors request, you should 1.) read the Lab Topic and appropriate sections of your text book, and 2) answer the Pre-Lab questions.

Outline

Introduction

Exercise 6.1 Catalase Activity

Exercise 6.2 Effect of Temperature on Enzyme Activity

Exercise 6.3 Effect of Concentration on Enzyme Activity

Exercise 6.4 Effect of pH on Enzyme Activity

Introduction

     Life as we know it is impossible without enzymes. The energy required by your muscles simply to open your lab manual would take years to accumulate without enzymes. Due to the presence of enzymes, the myriad chemical reactions occurring in your cells at this very moment are being completed in a fraction of a second rather than the years or even decades that would be otherwise required.

Enzymes are proteins that function as biological catalysts. A catalyst is a substance that lowers the amount of energy necessary for a chemical reaction to proceed.  

reactions may proceed from 100,000 to 10 million times faster than they would without the enzyme.

     The cell carries out many chemical reactions. All the chemical reactions that occur in a cell are collectively called metabolism. A possible chemical reaction can be                        indicated like this:                                   

            A + B                            C + D

          reactants       products

          In all chemical reactions, the reactants are molecules that undergo a change, which results in the products. The arrow stands for the change that produced the product(s). The number of reactants and products can vary; in the one you are studying today, a single reactant breaks down to two products.

          All the reactions that occur in a cell use an enzyme. In today's laboratory, you will be studying the action of the enzyme catalase. In an enzyme-catalyzed reaction, the reactant or reactants (the substances being acted upon) are called the substrate(s).          Enzymes are specific because they have a shape that accommodates the shape of  their substrates as a key fits a lock. Enzymatic reactions can be indicated like this:                      E + S                      ES                     E + P

In this reaction, E = enzyme, ES = enzyme-substrate complex, and P = product. Substrate molecules combine with enzyme molecules to form a temporary enzymesubstrate complex.  Products are formed and the enzyme molecule is released unchanged.  Thus the enzyme is not used up in the process and is capable of catalyzing the same reaction again and again.  

Figure 6.2  Action of an enzyme.

Look at Figure 6.2.  If all of the padlocks have the same interior shape (they are all the same substrate), the key (enzyme) can open any one, over and over again. A cell needs only a small amount of an enzyme because enzymes are used over and over.  Some enzymes can produce over a million product molecules per minute. 

          Two types of enzymatic reactions common to cells are shown in Figure 6.3 (below).  During degradation reactions, the substrate is broken down to the product(s), and during synthesis reactions, the substrates are joined to form a product.  Notice in each

Figure 6.3  The enzyme and substrate come together, and the reaction occurs on the surface of the enzyme at the active site.  The product leaves the enzyme, and then the enzyme can be used again.  a. During degradation, the substrate is broken down.  b. During synthesis, substrates combine to produce the product.  

case how the shape of the enzyme accommodates its substrate.  The location where the enzyme and substrate form an enzyme-substrate complex is called the active site because the reaction occurs there.  

Exercise 6.1 Catalase Activity

In the Experimental Procedures that follow, you will be working with the enzyme catalase. Catalase is present in cells, where it speeds the breakdown of the toxic chemical hydrogen peroxide to water and oxygen:

                                                             catalase        2H₂O₂              2H₂O  +  O₂

                                hydrogen peroxide             water    oxygen

What is the reactant in this reaction?_______What is the substrate for catalase?______ What are the products in this reaction?________and_________Bubbling occurs as the reaction proceeds. Why?_________________________________________________

Experimental Procedure: Catalase Activity

1.  With a wax pencil, label and mark three clean test tubes at the 1 cm and 5 cm levels.

Table 6.1 Catalase Activity

Conclusions

         Which tube showed the bubbling you expected?________Conclude why this tube 

      showed bubbling, and record your explanation in Table 6.1

         Which tubes are  a control?_________If this tube showed bubbling, what could            you conclude about your procedure? ___________Record your explanation in        Table 6.1 for this tube.

         Enzymes are specific; they speed only a reaction that contains their substrate.          Which tube exemplifies this characteristic of an enzyme?______Record your       explanation in Table 6.1 for this tube.

Exercise 6.2 Effect of Temperature on Enzyme Activity

In general, cold temperatures slow chemical reactions, and warm temperatures speed chemical reactions. Boiling, however, causes an enzyme to denature in a way that inactivates it.

Experimental Procedure: Effect of Temperature

Predict the activity you will see in tube 1 (cold), tube 2 (warm), tube 3 (boiled).

Tube 1.________________________________________________________________

Tube 2.________________________________________________________________

Tube 3.________________________________________________________________

With a wax pencil, label and mark three clean test tubes at the 1 cm and 5 cm levels.

1. Fill each tube to the first mark with catalase buffered at pH 7.0, the optimum pH for catalase.
2. Place tube 1 in a refrigerator or cold water bath, tube 2 in an incubator or warm water bath, and tube 3 in a boiling water bath. 

Complete the second column in Table 6.2. Wait 15 minutes.

3. As soon as you remove the tubes one at a time from the refrigerator, incubator, and boiling water, fill to the second mark with hydrogen peroxide.
4. Swirl well to mix, and wait 20 seconds.
5. Measure the height of the bubble column (in millimeters) in each tube, and record your results in Table 6.2.
6. Plot your results in Figure 6.4. Put temperature (^oC) on the X-axis and bubble column height (mm) on the Y-axis.

Table 6.2   Effect of Temperature

Conclusions           

 The amount of bubbling corresponds to the degree of enzyme activity. Explain in Table 6.2 the degree of enzyme activity per tube.

 What is your conclusion concerning the effect of temperature on enzyme activity? ___________________________________________________________________

___________________________________________________________________

_________________________________________________________________________                                   Figure 6.4 Effect of temperature on enzyme activity.

6.3 Effect of Concentration on Enzyme Activity

In general, a higher enzyme or substrate concentration results in faster enzyme activity-that is, the amount of product per unit time for any particular reaction will increase.

Experimental Procedure: Effect of Enzyme Concentration With a wax pencil, label and mark three clean test tubes. 

Tube 1          1.       Mark this tube at the 1 cm and 5 cm levels.

1. 2. Fill to the first mark with buffered catalase and to the second mark with                 hydrogen peroxide. Swirl well to mix, and wait 20 seconds.
   3. Measure the height of the bubble column (in millimeters), and record                       your results in Table 6.

Tube 2          1.       Mark this tube at the 2 cm and 6 cm levels.

1. 2. Fill to the first mark with buffered catalase and to the second mark with                 hydrogen peroxide. Swirl well to mix, and wait 20 seconds.
   3. Measure the height of the bubble column (in millimeters), and record                       your results in Table 6.3.

Tube 3     1.    Mark this tube at the 3 cm and 7 cm levels.

                    2.       Fill to the first mark with buffered catalase.  Fill to the second mark                   with hydrogen peroxide. Swirl well to mix, and wait at least 20 seconds.                      3.       Measure the height of the bubble column (in millimeters), and record                  your results in Table 6.3.

Table 6.3 Effect of Enzyme Concentration

Figure 6.5.  Effect of Enzyme concentration on Enzyme activity

Conclusions

 The amount of bubbling corresponds to the degree of enzyme activity.  Explain in Table 6.3 the degree of enzyme activity per tube.

 If unlimited time were allotted, would the results be the same in all tubes?_______

Explain why or why not._________________________________________________

 Would you expect similar results if the substrate concentration were varied in the same manner as the enzyme concentration?_______Why or why not?______________

Experiment 6.4 Effect of pH on Enzyme Activity

Each enzyme has a pH at which the speed of the reaction is optimum (occurs best). Any higher or lower pH affects the hydrogen bonding and structure of the enzyme, leading to reduced activity.

Experimental Procedure: Effect of pH

Caution: Hydrochloric acid (HCI) used to produce an acid pH is a strong, caustic acid, and sodium hydroxide (NaOH) used to produce a basic pH is

a strong, caustic base. Exercise care in using these chemicals, and follow your instructor's directions for disposal of tubes that contain these chemicals. If any acidic or basic solutions spill on your skin, rinse immediately with clear water.

With a wax pencil, label and mark three clean test tubes at the 1 cm, 3 cm, and 7 cm levels. Fill each tube to the 1 cm level with nonbuffered catalase.

Table 6.4 Effect of pH.

Figure 6.6 

Effect of pH on enzyme activity.        

Conclusions

 The amount of bubbling corresponds to the degree of enzyme activity. Explain in Table 6.4 the degree of enzyme activity per tube.

 The results of which tube in Table 6.1 could be used as a control for Table 6.4?____ 

Why could this tube be considered a control?__________________________________

Factors That Affect Enzyme Activity

In Table 6.5, summarize what you have learned about factors that affect the speed of an enzymatic reaction. For example, in general, what type of temperature promotes enzyme activity, and what type inhibits enzyme activity? Answer similarly for enzyme or substrate concentration and pH.

Table 6.5 Factors That Affect Enzyme Activity

Conclusions

Why does a warm temperature promote enzyme activity? ________________________

Why does increasing enzyme concentration promote enzyme activity?______________ 

Why does optimum pH promote enzyme activity?_____________________________

Laboratory Review 6

1. What happens at the active site of an enzyme?______________________________

_____________________________________________________________________

2. On the basis of the active site, explain why the following conditions speed a  chemical reaction:
   1. More enzyme______________________________________________________
   2. More substrate_____________________________________________________
3. Name three other conditions (other than the ones mentioned in question 2) that maximize enzymatic reactions.           a.

____________________________________________________________________  b.

____________________________________________________________________  c.

____________________________________________________________________

4. Explain the necessity for each of the three conditions you listed in question 3.   a.

____________________________________________________________________  b.

____________________________________________________________________  c.

____________________________________________________________________

5. Lipase is a digestive enzyme that digests fat droplets in the basic conditions (NaHCO₃ is present) of the small intestine. Indicate which of the following test tubes  would show digestion following incubation at 37^oC, and explain why the others would not.

         Tube 1: Water, fat droplets

____________________________________________________________________

         Tube 2: Water, fat droplets, lipase

____________________________________________________________________           Tube 3: Water, fat droplets, lipase, NaHCO₃

____________________________________________________________________

         Tube 4: Water, lipase, NaHCO₃

____________________________________________________________________

6. Fats are digested to fatty acids and glycerol. As the reaction described in question 5 proceeds, the solution will become what type pH?___________ Why?

____________________________________________________________________

7. Given the following reaction:

                                                           catalase                            2 H₂O₂          2H₂O  +  O₂

                                            hydrogen            water oxygen                                       peroxide

1. 1. 1. Which substance is the substrate?______________________________________

1. 1. 2. Which substance is the enzyme?_______________________________________

1. 1. 3. Which substances are the end products?_________________________________

1. 1. 4. Is this a synthetic or degradative reaction?_______________________________

How do you know?___________________________________________________

PRE-LAB QUESTIONS Enzymes

Circle the correct answer

1. Enzymes are

(a) biological catalysts (b) agents that speed up cellular reactions (c) proteins

(d) all of the above

2. Enzymes function by (a) being consumed (used up) in the reaction

2. lowering the activation energy of a reaction
3. combining with otherwise toxic substances in the cell  (d) adding heat to the cell to speed up the reaction

3.  The substance that an enzyme combines with is (a) another enzyme

2. a cofactor
3. a coenzyme
4. the substrate

4. Enzyme specificity refers to the (a)  need for cofactors for some enzymes to function

(b)  fact that enzymes catalyze one particular substrate or a small number of structurally similar substrates (c)  effect of temperature on enzyme

activity

(d)  effect of pH on enzyme activity

5. When an enzyme becomes denatured, it

1. increases in effectiveness
2. loses its requirement for a cofactor
3. forms an enzyme-substrate complex
4. loses its ability to function

6. An enzyme may lose its ability to function because of (a)  excessively high temperatures (b)  a change in its three-dimensional structure

3. a large change in the pH of the

          environment

4. all of the above

7. pH is a measure of

1. an enzyme's effectiveness
2. enzyme concentration
3. the hydrogen-ion concentration
4. none of the above

8.  Catalase

(a)  is an enzyme found in browning fruit (b)  catalyzes the breakdown of hydrogen peroxide to water (c)  has as its substrate NaCO3 (d)  is a substance that encourages the

growth of microorganisms 

9. The bubbling height used in the experiments of this exercise (a)  is a consequence of production of oxygen and water

2. is an index (shows the amount) of enzyme activity
3. may differ depending on the pH, temperature, or amount of substrate, respectively (d)  is all of the above