Laboratory 7: Enzyme Activity

1. Learning Objectives

   By the end of this laboratory exercise, you should be able to:

   • Describe what enzymes are and explain their role in biological systems.

   • Distinguish among enzymes, substrates, and products.

   • Explain how an enzyme’s active site contributes to its function.

   • Demonstrate enzyme specificity by comparing how an enzyme responds to different substrates.

   • Investigate how temperature, pH, and enzyme concentration influence enzyme activity.

   • Interpret experimental outcomes using observable indicators such as gas or bubble production.

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   Key Terms

   Term	Definition
   Enzyme	A biological catalyst—typically a protein—that increases the rate of chemical reactions without being consumed.
   Substrate	The molecule that an enzyme acts upon.
   Product	The substance(s) produced after an enzyme-catalyzed reaction.
   Active Site	The region of an enzyme where substrate binding and catalysis occur.
   Enzyme Specificity	The characteristic of enzymes that allows them to catalyze only certain reactions or interact with specific substrates.
   Catalase Reaction	The breakdown of hydrogen peroxide into water and oxygen.

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   Introduction

   Enzymes are essential to life. They allow chemical reactions to occur rapidly under the mild temperatures and pH conditions found in living organisms. Without enzymes, most biochemical reactions would proceed too slowly to sustain life.

   Each enzyme interacts with a particular substrate. When the substrate binds to the enzyme’s active site, an enzyme–substrate complex forms. The structural compatibility between the enzyme and substrate—often compared to a lock fitting a key—helps ensure that only specific reactions occur.

   Several factors influence enzyme activity:

   • Temperature affects molecular movement and can alter enzyme structure.

   • pH changes the electrical charges and shape of the active site.

   • Enzyme concentration determines how many catalytic molecules are available.

   • Substrate identity controls whether the enzyme can carry out the reaction.

   In this lab, you will study these factors using the enzyme to investigate its activity under different environmental conditions.

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   Experimental Procedures

   Experiment 1 – Enzyme–Substrate Specificity

   Purpose

   To determine whether the enzyme reacts differently when exposed to similar but non-identical substrates.

   Materials

   • Yeast suspension

   • 10% glucose solution

   • 10% galactose solution

   • Two J-tubes

   • Color-coded pipettes

   • 37°C water bath

   • Ruler

   Procedure

   1. Use a separate pipette for each solution to avoid contamination.

   2. Add 15 mL of yeast suspension to each of the two J-tubes.

   3. Add 15 mL of 10% glucose to one tube and 15 mL of 10% galactose to the other.

   4. Tilt each J-tube backward to allow the solution to completely fill the side arm.

      • If the side arm does not fill, repeat the setup.

   5. Place both tubes in the 37°C water bath for 1 hour without disturbing them.

   6. Measure the liquid displacement (mm) in the side arm, representing CO₂ production.

   Data Table 1. Substrate Specificity

   Substrate	Liquid Displacement (mm)	Gas Produced	Observation / Interpretation
   Glucose
   Galactose

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   Experiment 2 – Factors Affecting Enzyme Activity

   This series of experiments examines how substrate identity, temperature, enzyme concentration, and pH influence the activity of the enzyme when reacting with hydrogen peroxide.

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   Part A – Effect of Enzyme Presence and Substrate Type

   Materials

   • Hydrogen peroxide

   • Enzyme solution

   • Sucrose solution

   • Water

   • Test tubes

   • Sharpie marker

   • Ruler

   Procedure

   1. Label three test tubes and mark each at the 1 cm and 5 cm levels.

   2. Prepare the tubes: Add enzyme to the first mark. Add hydrogen peroxide to the second mark.

   Tube	Contents	Expected Reaction
   1	Enzyme + Hydrogen Peroxide	Strong bubbling (oxygen produced)
   2	Water + Hydrogen Peroxide	No bubbling
   3	Enzyme + Sucrose	No bubbling

   3. Swirl gently.

   4. After 20 seconds, measure bubble height (mm).

   Data Table 2. Evidence of Enzyme Activity

   Tube	Contents	Bubble Height (mm)	Explanation
   1	Enzyme + Hydrogen Peroxide
   2	Water + Hydrogen Peroxide
   3	Enzyme + Sucrose

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   Part B – Effect of Temperature on Enzyme Activity

   Materials

   • Three test tubes

   • Ice bath (0°C)

   • Warm water bath (37°C)

   • Boiling water bath (100°C)

   • Enzyme solution

   • Hydrogen peroxide

   • Ruler

   Procedure

   1. Label and mark all tubes at 1 cm and 5 cm.

   2. Add enzyme to the 1 cm mark.

   3. Place:

      • Tube 1 in ice (0°C)

      • Tube 2 in 37°C water bath

      • Tube 3 in boiling water (100°C)
        for 15 minutes.

   4. Add hydrogen peroxide to the 5 cm mark.

   5. Swirl and measure bubble height after 20 seconds.

   Data Table 3. Temperature and Enzyme Activity

   Tube	Temperature Condition	Bubble Height (mm)	Explanation
   1	Ice (0°C)
   2	Warm (37°C)
   3	Boiling (100°C)

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   Part C – Effect of Enzyme Concentration

   Procedure

   1. Label tubes 1–3.

   2. Mark:

      • Tube 1 at 1 cm and 5 cm

      • Tube 2 at 2 cm and 6 cm

      • Tube 3 at 3 cm and 7 cm

   3. Add enzyme to the first mark.

   4. Add hydrogen peroxide to the second mark.

   5. Swirl gently.

   6. Measure bubble height after 20 seconds.

   Data Table 4. Enzyme Concentration and Reaction Rate

   Tube	Amount of Enzyme (cm)	Bubble Height (mm)	Explanation
   1	1
   2	2
   3	3

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   Part D – Effect of pH

   Materials

   • pH buffers (3, 7, 10)

   • Enzyme solution

   • Hydrogen peroxide

   • Test tubes

   • Ruler

   Procedure

   1. Mark tubes at 1 cm, 3 cm, and 7 cm.

   2. Add enzyme to the 1 cm mark.

   3. Add buffer to the 3 cm mark:

      • Tube 1: pH 3

      • Tube 2: pH 7

      • Tube 3: pH 10

   4. Add hydrogen peroxide to the 7 cm mark.

   5. Allow to sit 1 minute, swirl gently, and measure bubble height after 20 seconds.

   Data Table 5. pH Effects on Enzyme Activity

   Tube	pH	Bubble Height (mm)	Explanation
   1	3
   2	7
   3	10

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   Analysis and Discussion

   Provide complete responses in the spaces below.

   1. Define the following terms: enzyme, substrate, product, active site, and enzyme specificity.

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   2. Which substrate—glucose or galactose—supported fermentation? Explain your reasoning.

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   3. In Experiment 2 (Part A), which tube produced the greatest amount of bubbling? Why?

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   4. Describe what happened to the enzyme’s activity when the temperature reached boiling.

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   5. How did changing enzyme concentration affect the reaction rate?

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   6. At which pH level did the enzyme show the greatest activity? Explain.

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   7. Why are enzymes sensitive to changes in temperature and pH?

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   8. What gas was released during the enzyme reaction in experiment 2?

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   9. What happens when an enzyme becomes denatured?

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   10. Why are enzymes essential to living organisms?

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Licenses and Attribution

CC Licensed Content, Original:

Other Licensed Content Included:

• Adapted with permission from BioScience I Laboratory Manual by Jamie Colson-Moon and Denise Bristol. Per request from these original authors, this content is now licensed (CC BY-NC-SA).