EXERCISE 5

ENZYMATIC ACTIVITY

I. INTRODUCTION

Enzymes, typically proteins, are macromolecular biological catalysts that lower the
activation energy of biochemical reactions, enabling these reactions to occur at cellular
temperatures. Each enzyme's specificity arises from its unique three-dimensional structure,
which determines its function. Like other catalysts, enzymes accelerate reactions without
being consumed in the process. They achieve this by binding to specific reactants known as
substrates. An enzyme-catalyzed reaction begins when the substrate binds to the enzyme's
active site, forming an enzyme-substrate complex. The substrate is held in the active site by
weak interactions such as hydrogen bonds and ionic bonds. Upon substrate binding, the
enzyme undergoes slight structural changes, aligning the active site to better interact with the
substrate, a model known as the induced fit model. This optimizes the enzyme's catalytic
efficiency. Side chains of amino acids in the active site facilitate the conversion of the substrate
into the product, and once the reaction is complete, the product is released, and the enzyme
returns to its original form, ready to catalyze another reaction.

Metabolic pathways rely on enzymes to catalyze individual steps, with enzymes capable
of catalyzing over 5,000 types of biochemical reactions. While most enzymes are proteins,
some catalytic RNA molecules, called ribozymes, also exist. Enzyme activity can be
modulated by other molecules; inhibitors decrease enzyme activity, while activators increase
it. An enzyme’s activity is also influenced by temperature and pH, with activity decreasing
significantly outside its optimal range. Enzymes have numerous commercial and practical
applications, such as in the industrial synthesis of antibiotics and in household products like
biological washing powders and meat tenderizers.

Rate of enzyme activity = distance / time where distance is the depth of the hydrogen
peroxide in mm and time measured in seconds.

In this laboratory, we will investigate the effect of temperature, concentration, and pH on

the activity of the enzyme catalase (enzyme present in potato or yeast, Saccharomyces
cerevisiae), providing insights into how environmental conditions influence enzyme function.

II. OBJECTIVES

1. To explain the procedures for determining the catalase activity

2. To explain the effects of temperature and pH on enzymatic reactions
3. To explain the effects of different enzyme and substrate concentrations on the rate of
enzymatic reactions
4. To describe the effects of the above factors on catalase activity

III. MATERIALS

Fresh potato Test tubes Water bath (37°C and 97°C)

Hydrogen peroxide Test tube rack Refrigerator / Ice bath
Distilled water Blender Graduated cylinder
pH solution (pH 3,5,7,9,12) Cheese cloth/filter Pipette
Sucrose Thermometer Timer

IV. PROCEDURE
 ▪ Catalase Enzyme Preparation:
1. Potato extract
a. Weigh 50 g of peeled fresh potato.
b. Slice into small cubes and homogenized in a blender by initially adding 50 mL
of cold distilled water.
c. Filter the mixture through a cheese cloth and bring to a total volume of 100 mL.

2. Yeast
a. Weigh 2 g of yeast
b. Add 100 mL of warm distilled water and mix well.
c. Sit for at least 3 minutes.

A. Experimental Procedure (Negative and Positive Controls)

3. Obtain and label the three (3) 15 ml conical plastic tubes as tube 1, tube 2, and
tube 3.
4. In tube 1; add 1 ml of catalase enzyme and 4 ml of hydrogen peroxide.
5. In tube 2; add 1 ml of water and 4 ml of hydrogen peroxide
6. In tube 3; add 1 ml of catalase enzyme and 4 ml of sucrose solution,
7. Swirl well to mix and wait at least 20 seconds.
8. Measure the height of the bubble column in each tube and calculate the rate of
enzyme activity. Record the result in Table 5.1.

B. Effect of temperature on enzyme activity

1. Before you begin with the actual experiment, write down in your own words the
hypothesis for this experiment
2. Obtain and label three tubes.
3. Add 1 ml of catalase enzyme to each tube.
4. Place tube 1 in the refrigerator (~4°C) or ice bath, tube 2 in a 37°C (Celsius) heat
block, and tube 3 in a 97°C heat block for 15 minutes.
5. Remove the tubes with the catalase enzyme from the refrigerator and heat blocks
and immediately add 4 ml hydrogen peroxide to each tube.
6. Swirl well to mix and wait at least 20 seconds.
7. Measure the height of the bubble column in each tube and calculate the rate of
enzyme activity. Record the result in Table 5.2.

C. Effect of concentration on enzyme activity

1. Before you begin with the actual experiment, write down in your own words the
hypothesis for this experiment.
2. Obtain and label five tubes.
3. In tube 1, add 1 ml of water; in tube 2, add 1 ml of catalase enzyme; in tube 3, add
3 ml of catalase enzyme; in tube 4, add 5 ml of catalase enzyme; and in tube 5,
add 10 ml of catalase enzyme.
4. To each tube, add 4 ml of hydrogen peroxide, swirl well to mix and wait at least 20
seconds.
5. Measure the height of the bubble column in each tube and calculate the rate of
enzyme activity. Record the result in Table 5.3.

D. Effect of pH on enzyme activity

1. Before you begin with the actual experiment, write down in your own words the
hypothesis for this experiment.
2. Obtain 6 tubes and label each tube with a number from 1 to 6.
3. Place the tubes from left (tube #1) to right (tube #6) in the first row of a test tube
rack.
4. Add to 1 ml of catalase enzyme to each tube.
 5. Add 2 ml of water to tube 1.
6. Add 2 ml of pH buffer 3 to tube 2.
7. Add 2 ml of pH buffer 5 to tube 3.
8. Add 2 ml of pH buffer 7 to tube 4.
9. Add 2 ml of pH buffer 9 to tube 5.
10. Add 2 ml of pH buffer 12 to tube 6.
11. Add 4 ml of hydrogen peroxide to each of the six tubes.
12. Swirl each tube well to mix and wait at least 20 seconds.
13. Measure the height of the bubble column in each tube and calculate the rate of
enzyme activity. Record the result in Table 5.4.

V. RESULTS AND OBSERVATIONS

Record your results in the answer sheet

REFERENCES

Bhatt, K., Davis, A. M., & Ahmed, S. A. (2019). Heating up Enzyme Kinetics: A Safe,
Inexpensive & Quantitative Inquiry Activity. The American Biology Teacher, 81(3), 187-
192.
Latourelle, S. M., Elwess, N. L., & Ryan, A. B. (2020). Tried and true but something new:
analyzing the enzymatic activity of catalase. Journal of Biological Education, 54(5),
540-547.

Vartak, R., Ronad, A., & Ghanekar, V. (2013). Enzyme assay: An investigative approach to
enhance science process skills. Journal of Biological Education, 47(4), 253-257.

Laboratory Manual for Biology Course. Available at

https://myrcc.rcc.mass.edu/ICS/103_lab_manual/enzymes.html

Experiment on Enzyme. Available at

https://laney.edu/cheli-fossum/wp-content/uploads/sites/210/2012/01/10-Enzymes.pdf
Name: Rating:
Laboratory Schedule: Date Performed:
Instructor:

EXERCISE 4

ENZYMATIC ACTIVITY

V. RESULTS AND OBSERVATIONS

Procedure A.

Table 5. 1. Positive and Negative Controls

Heights of the Rate of Enzyme Observation

Tube no. Time (seconds) Activity
bubbles (mm)
1
2
3

Which tube is experimental, negative and positive control? What is your basis?

Procedure B.

Table 5. 2. Effect of temperature on enzyme activity.

Heights of the Rate of Enzyme Observation

Tube no. Time (seconds) Activity
bubbles (mm)
1
2
3

Hypothesis:

Do the data support or contradict your hypothesis? Explain.

 Procedure C.

Table 5. 3. Effect of concentration on enzyme activity.

Heights of the Rate of Enzyme Observation

Tube no. Time (seconds) Activity
bubbles (mm)
1
2
3
4
5

Hypothesis:

Do the data support or contradict your hypothesis? Explain.

Procedure D.

Table 5. 4. Effect of pH on enzyme activity.

Heights of the Rate of Enzyme Observation

Tube no. Time (seconds) Activity
bubbles (mm)
1
2
3
4
5
6

Hypothesis:

Do the data support or contradict your hypothesis? Explain.

VI. CONCLUSION:
QUESTIONS:

1. What is the role of catalase in the reaction with hydrogen peroxide? Describe the
chemical reaction that takes place.

2. How does temperature affect the activity of catalase? Based on your observations,
explain what happens to catalase activity at 4°C, 37°C, and 97°C.

3. Why is it important to test different concentrations of enzyme extract in this experiment?

What trends did you observe regarding enzyme concentration and reaction rate?

4. Describe how pH levels influence the activity of catalase. Which pH level resulted in
the highest activity, and why might this be the case?

5. Explain why there was no reaction when catalase was added to the sucrose solution.
What does this tell you about the specificity of enzymes?

6. Ectothermic organisms have body temperatures that vary with the temperature of their
surroundings. Discuss the effect this variation might have on the functioning of
enzymes in these organisms. Suggest some ways ectothermic organisms might cope
with this problem.

REFERENCES