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LABORATORY 2 - Enzyme Catalysis

LABORATORY 2: ENZYME CATALYSIS


OVERVIEW
In this laboratory you will learn how to determine the quantity of a substance in a solution via the titration method and then calculate the rate of conversion of hydrogen peroxide (H2O2) to water and oxygen gas by the enzyme catalase.

OBJECTIVES
Before doing this laboratory you should understand: - the general functions and activities of enzymes - the relationship between structure and function of enzymes - the concept of initial reaction rates of enzymes - how the concept of free energy relates to enzyme activity - how pH relates to enzyme activity - that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions - the concept of titration After doing the laboratory you should be able to: - measure the effects of changes of temperature, pH, enzyme concentration, and substrate concentration on reaction rates of enzyme-catalyzed reactions - explain how environmental factors affect the rate of enzyme-catalyzed reactions

INTRODUCTION
Enzymes are proteins produced by living cells that act as catalysts in biochemical reactions. A catalyst increases the rate of a chemical reaction by lowering the activation energy (Biology by Campbell, Reece & Mitchell, 5th ed., pp. 97-99). One benefit of enzyme activity is that the cell can carry out complex chemical activities at relatively low temperatures. In an enzyme-catalyzed reaction, the substance to be acted upon, the substrate (S), binds reversibly to the active site of the enzyme (E). One result of this temporary union is a reduction in the energy required to activate the reaction of the substrate molecule so that the products (P) of the reaction are formed.

E + S ES E + P
Note that the enzyme is not changed or used up in the reaction; it can recycle to break down additional substrate molecules. Each enzyme is specific for a particular reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The active site of each enzyme is therefore unique so that any substance that blocks or changes the shape of the active site affects the activity of the enzyme. An enzyme whose shape is changed significantly so that it no longer functions effectively as a catalyst is said to be denatured. A description of several ways enzyme action may be affected follows.

1. Salt concentration. If the salt concentration is very low or zero, charged amino acid side chains of the enzyme molecules will attract each other. The enzyme will denature and form an inactive precipitate. If, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked, new interactions will occur, and again the enzyme will precipitate. An intermediate salt concentration such as that of blood (0.9%) or cytoplasm is the optimum for most enzymes. -1HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

2. pH. pH is a logarithmic scale that measures the concentration of protons (H ) in a solution. The scale runs + + from 0 to 14 with 0 = high concentration of H (very acidic) and 14 = low [H ] (very basic). A pH of 7 is neutral. Amino acid side chains contain groups such as -COOH and -NH that readily gain or lose H+ ions.
2

As the pH is lowered an enzyme will tend to gain H+ ions. Eventually enough side chains may be affected so that the enzymes shape is disrupted. Likewise, as the pH is raised, the enzyme will lose H+ ions, and may eventually lose its active shape. Many enzymes work optimally in the neutral pH range and are denatured at either extremely high or low pH. Some enzymes, such as those in the human stomach, where the pH is very low, will act optimally at a low pH. A buffer is a compound that will gain or lose H+ ions so that the pH changes very little. 3. Temperature. All chemical reactions speed up as the temperature is raised. As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme-catalyzed reaction is raised still further, a temperature optimum is reached; above this point, the kinetic energy of the enzyme and water molecules is so great that the structure of the enzyme starts to be disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of denaturing more and more enzyme molecules. Many proteins are denatured by temperatures around 40-50C, although a few even withstand being boiled. 4. Small molecules. Many molecules other than the substrate may interact with an enzyme. If such a molecule increases the rate of the reaction, it is an activator. If it decreases the reaction rate, it is an inhibitor. The cell can use these molecules to regulate how fast an enzyme acts. Any substance that tends to unfold the enzyme, such as an organic solvent or detergent, will act as an inhibitor. Some inhibitors act by reducing the disulfide (-S-S-) bridges that stabilize the enzymes structure. Many inhibitors act by reacting with side chains in or near the active site to change or block it. Others may damage or remove the prosthetic group. Many wellknown poisons such as potassium cyanide and curare are enzyme inhibitors that interfere with the active site of a critical enzyme.

The enzyme used in this lab, catalase, has four polypeptide chains, each composed of more than five hundred amino acid residues. The enzyme occurs universally in aerobic organisms. One function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide (H2O2) formed as a by-product of some metabolic processes. Catalase may also take part in some of the many oxidation-reduction reactions occurring in all cells. The primary reaction catalyzed by catalase is the decomposition of H2O2 to form water and oxygen.

2 H2O2 2 H2O + O2 (gas)


This reaction occurs spontaneously, but not at a very rapid rate. Catalase speeds up the reaction considerably. In this experiment, a rate for this reaction will be determined. The rate of a chemical reaction may be studied in a number of ways, including the following: 1. Measuring the rate of disappearance of substrate; in the example, H2O2 2. Measuring the rate of appearance of product; in this case, O2, which is given off as a gas. 3. Measuring the heat released (or used) during the reaction.

-2HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

Each chemical reaction has a characteristic amount of heat that it either gives off or absorbs per mole of product produced. Therefore, the amount of heat generated or absorbed by the reaction will be proportional both to the amount of product formed and the amount of reactants consumed. If the amount of product formed is measured at 30-second intervals and this quantity is plotted on a graph, a curve like the one that follows is obtained. Figure 2.1: Enzyme Activity

Product (moles)

40 30 20 10 1 2 3 4 5 6 7 Time (minutes) 8

Observe the solid line for this reaction. At time 0 there is no product. After 30 seconds, five mols have been formed; after one minute, 10; after two minutes, 20. The rate of this reaction could be given as 10 mols of product formed per minute for this initial period. Note, however, by the fourth minute, only 5 additional mols of product have been formed. During the first three minutes, the rate is constant. From the third minute through the eighth minute, the rate is changing it is decreasing; the amount of product formed in each minute interval is less than in the preceding minute. From the seventh minute onward, very little product is formed. To compare the kinetics of one reaction with another, a common reference point is needed. For example, suppose you wanted to compare the effectiveness of catalase obtained from potato with that of catalase obtained from liver. Would you want to compare the two reactions during the first few minutes, when the rate is constant, or later, when the rate is changing? It is best to compare the reactions when the rates are constant. In the first few minutes of an enzymatic reaction such as this, the number of substrate molecules is usually so large compared to the number of enzyme molecules that changing the substrate concentration does not (for a short period at least) affect the number of successful collisions between substrate and enzyme. During this early period, the enzyme is acting on substrate molecules at a constant rate, and the enzyme is said to be saturated. The slope of the graph line during this early period is called the initial rate of the reaction. The initial rate of an enzyme-catalyzed reaction is determined by the characteristics of the enzyme molecule. It is always the same for an enzyme and its substrate as long as temperature, pH, and salinity are constant and the substrate is present in excess. The rate of the reaction, therefore, is the slope of the linear portion of the curve. To determine a rate, pick any two points on the straight-line portion of the curve, divide the difference in the amount of product formed between these two points by the difference in time between them. The result will be the rate of the reaction, which can be expressed as mol per second. The rate then is:

moles2 moles1
t 2 t1

In the illustration of Figure 2.1, the rate between 2 and 3 minutes is calculated:

30 20 10 = = 0.17 moles / s 180 120 60


-3HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

EXERCISE 2A: TEST OF CATALASE ACTIVITY


MATERIALS ~ 1.5% (0.44-M) H2O2 solution ~ catalase solution ~ potato or liver tissue (1 cm3) ~ three 50 mL glass beakers ~ boiling water bath PROCEDURE only for question 1: (Be sure to answer questions 1, 2 and 3 for your lab writeup) 1. To observe the reaction to be studied, transfer 10 mL of H2O2 solution into a 50 mL glass beaker and add 1 mL of the catalase solution. a. What is the enzyme in this reaction? _________________________________________ b. What is the substrate in this reaction? ________________________________________ c. What are the products in this reaction? ________________________________________

2.

To demonstrate the effect of boiling on enzymatic activity, transfer 5 mL of purified catalase extract to a test tube and place it in a boiling water bath for 5 minutes. Transfer 10 mL of H2O2 into a 50 mL glass beaker and add 1 mL of a boiled catalase solution. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference.

3.

To demonstrate the presence of catalase in living tissue, cut 1 cm3 of potato or liver, macerate it, and transfer it into a 50-mL glass beaker containing 10 mL of 1.5% H2O2 . What do you observe? What do you think would happen if the potato or liver was boiled before being added to the H2O2 ?

-4HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

EXERCISES 2B & 2C:


MATERIALS ~ 2% (0.13-M) KMnO4 ~ 1.5% (0.44-M) H2O2 ~ 1.0-M H2SO4 ~ catalase solution ~ distilled H2O ~ burette ~ burette holder ~ ring stand ~ six 50 mL glass beaker/8 oz. plastic cups ~ six small plastic reaction cups (2 oz. plastic cups) ~ disposable pipettes/droppers ~ graduated cylinders ~ GLOVES & GOGGLES

*BEFORE STARTING EX. 2B & 2C, REVIEW THE TITRATION PROCEDURE BELOW; YOU WILL BE UTILIZING THIS PROCEDURE IN BOTH EXERCISES* DO NOT BEGIN WITH THIS PROCEDURE - IT SIMPLY PROVIDES AN OVERVIEW OF WHAT YOU ARE ABOUT TO DO!

GENERAL TITRATION PROCEDURE TO BE USED IN EXERCISES 2B and 2C : The amount of hydrogen peroxide (H2O2) will be measured using a titration with potassium permanganate. The general procedure for this titration is as follows: 1. A purified catalase is mixed with substrate (H2O2) in a beaker. The enzyme catalyzes the conversion of H2O2 to H2O and O2 (gas). Before all the H2O2 is converted to H2O and O2, the reaction is stopped by adding sulfuric acid (H2SO4). The sulfuric acid lowers the pH, denatures the enzyme, and thereby stops the enzymes catalytic activity. After the reaction is stopped, the amount of H2O2 remaining in the beaker is measured. To assay (measure) this quantity, potassium permanganate (KMnO4) is used. Potassium permanganate, in the presence of H2O2 and H2SO4, reacts as follows:

2.

3.

5 H2O2 + 2KMnO4 + 3 H2SO4> K2SO4 + 2Mn SO4 + 8 H2O + 5 O2


Note that H2O2 is a reactant for this reaction. Once all the H2O2 has reacted, any more KMnO4 added will be in excess and will not be decomposed. The addition of excess KMnO4 causes the solution to have a permanent pink or brown color. Therefore, the amount of H2O2 remaining is determined by adding KMnO4 until the whole mixture stays a faint pink or brown, permanently. Add no more KMnO4 after this point (also known as the end point). The amount of KMnO4 added is a proportional measure of the amount of H2O2 remaining (2 molecules of KMnO4 reacts with 5 molecules of H2O2 as shown in the equation).

-5HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

Note: Handle KMnO4 with care. Avoid contact with skin and eyes.

-6HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

SET-UP:
Every two groups will share a buret set-up (but each group will have their own buret). Figure 2.2: General example:

group #1s buret

group #2s buret

1. Fold up a piece of Whatman #1 filter paper as demonstrated by your teacher. 2. Insert the cone-shaped filter paper into a funnel. 3. Using this funnel set-up, fill your burette with approximately 15 mL of KMnO4. 4. Allow approximately 1 mL of KMnO4 to run out into a "throw-away container.

Now you are ready to proceed with Exercise B.

**WEAR GOGGLES & GLOVES THROUGHOUT THIS LAB (i.e., until you have finished cleaning up!)

-7HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

EXERCISE 2B: THE BASELINE ASSAY


To determine the amount of H2O2 initially present in a solution, one needs to perform all the steps of the procedure without adding catalase (enzyme) to the reaction mixture. This amount is known as the baseline. In any series of experiments, a baseline should be established first. 1. 2. 3. 4. 5. Put 10 mL of H2O2 in a beaker or 8oz. plastic cup. Add 1 mL of dH2O. Add 10 mL of H2SO4 (1.0 M). USE EXTREME CARE IN HANDLING ACIDS. Your teacher will instruct you about the proper safety procedures for handling hazardous materials. Mix well. Remove only 5 mL into the small 2oz. cup and assay for the amount of H2O2 present: a. Now record the initial reading in Table 2.1. [why do you need to let 1 mL run out before starting the titration?] b. Place your sample beaker beneath the burette and over a piece of white paper. c. Add KMnO4 one drop at a time [why is this important?] to the sample beaker until a persistent pink or brown color is obtained and record the final reading. Remember to swirl the solution gently after adding each drop. Table 2.1 Initial reading of burette (mL) Final reading of burette (mL) Total volume KMnO4 used (mL)

To calculate the baseline, you need to calculate the moles of KMnO4 used in the titration. (Hint: What is the definition of molarity?) Using this number, you can calculate the amount of H2O2 that will react with this amount of KMnO4. (Hint: Look at the equation on p.5)

Show your calculations below (SHOW UNITS):

-8HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

EXERCISE 2C: AN ENZYME-CATALYZED RATE OF H2O2 DECOMPOSITION


In this experiment, you will determine the rate at which a H2O2 solution decomposes when catalyzed by purified catalase extract. To do this, you should determine how much H2O2 has been consumed after 10, 30, 60, 120, 180, 240, 300 seconds.

Each time, remove only 5 mL and assay for the amount of H2O2 remaining. Use the burette to
add KMnO4, a drop at a time, to the solution until a persistent pink or brown color is obtained. Should the end point be overshot, there is sufficient sample left to repeat the titration. Do the entire lab is completed. Record your results in Table 2.2 and graph.

not discard any of the sample until

10 seconds 1. 2. 3. 4. 30 seconds 1. 2. 3. 4.

Put 10 mL of H2O2 in a 50-mL glass beaker. Add 1 mL catalase extract. Swirl gently for 10 seconds. At 10 seconds, add 10 mL of sulfuric acid (1.0 M).

Put 10 mL of H2O2 in a 50-mL glass beaker. Add 1 mL catalase extract. Swirl gently for 30 seconds. At 30 seconds, add 10 mL of sulfuric acid (1.0 M).

60, 120, 180, 240, 300 seconds Each time, repeat steps 1 through 4 as above, except allow the reactions to proceed for 60, 120, and 180, 240, 300 seconds, respectively. *To use lab time more efficiently, set up these last 5 reaction cups at the same time, and do them together. Be sure to stop each reaction at the proper time.

Table 2.2 Baseline: ___________ 10 sec A. Initial reading (_____) B. Final reading (_______) C. Volume of KMnO4 used (_______) D. Moles of KMnO4 titrated E. Moles of H2O2 titrated F. Total amount of H2O2 consumed 30 sec 60 sec 120 sec 180 sec 240 sec 300 sec

-9HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

NOTE: F is the difference in the amount of H2O2 that was present before and after the enzyme-catalyzed reaction took place. (i.e. it is the amount of H2O2 consumed during the timed reaction.)

- 10 HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

ANALYSIS OF RESULTS
Graph the amount of H2O2 consumed versus the amount of time the reaction was allowed to take place. For this graph, you will need to determine the following: a. the independent variable? ______________________ b. the dependent variable? ________________________ (label the horizontal (X) axis) (label the vertical (Y) axis)

Title: ________________________________________________________________________

1. What is the hypothesis being tested in this experiment?

2. What is the variable factor in this experiment?

- 11 HHS A.P. Biology - Laboratory Manual

LABORATORY 2 - Enzyme Catalysis

3. What serves as a control in this experiment? Why is this control necessary?

4. From the formula described earlier, determine the initial rate of the reaction and the rates between each of the time points. (SHOW CALCULATIONS for one interval) Time Intervals (seconds) 30 - 60 60 - 120

Initial (0-10)

10 - 30

120 - 180

** Put the reaction rates (in mmol H2O2/sec) in the second row.

5. When is the rate the highest? Why?

6. When is the rate the lowest? For what reasons is the rate low?

7. Based on facts related to enzyme structure and chemistry, explain the inhibiting effect of sulfuric acid on the function of catalase.

8. Predict the effect lowering the temperature would have on the rate of enzyme activity. Explain.

- 12 HHS A.P. Biology - Laboratory Manual