1.7: Enzyme Function

In every living cell, many chemical reactions are performed. In chemical reactions, the reactants are molecules that undergo a change, which results in the products. The arrow indicates the direction of the reaction as the reactants proceed to be transformed into the product(s). As you can see in the figure below, the number of reactants and products can vary, but the number of atoms is the same on both sides of the arrow. During synthesis reactions (Example 1), the reactants are joined to form a product. During decomposition reactions (Example 2), the reactant is broken down into products. During exchange reactions (Example 3) both decomposition and synthesis take place. The decomposition of two reactants is followed by an exchange and synthesis of two new compounds from them.

Example 1 (Synthesis Reaction): \(\ce{A + B → AB}\)

\[\ce{2H2 + O2 → 2 H2O}\]

Example 2 (Decomposition Reaction): \(\ce{AB → A + B}\)

\[\ce{2H2O → 2 H2 + O2}\]

Example 3 (Exchange Reaction): \(\ce{A + BC → AC + B}\)

\[\ce{2 Na + 2 H2O → 2 NaOH + H2}\]

Enzymes are very efficient catalysts for biochemical reactions. These catalysts speed up reactions by providing an alternative reaction pathway that requires a lower activation energy needed for a reaction to get started. The lower the activation energy for a reaction, the faster the rate at which it will proceed. Enzymes only lower the activation energy, but do not change the difference in energy levels between the reactants and the products. They work by binding to the reactants and converting them to a different compound (the product). Enzymes are specific to a type of reactant, and therefore can catalyze only one type of reaction. This enzyme specificity is the result of the particular shape of the enzyme that only permits binding to one type of reactant, much like a key fits into a lock (Figure 2). The reactants in an enzymatic chemical reaction are called substrates. Notice how the shape of the enzyme fits its substrate. The location where the enzyme binds a substrate is called the active site because the reaction occurs here. At the end of the reaction, the product is released, and the enzyme can then bind to more substrate. Thus, an enzyme can perform the reaction over and over again, as long as the substrate is present.

Figure 2: Enzymes bind a specific substrate and convert it into the products. The products are released, and the enzyme is unchanged. (CC By 4.0; OpenStax)

Most enzymes are complex proteins that function most efficiently within a specific range of temperature and pH. Extremes in temperature or pH will denature the enzyme by permanently altering its chemical structure. Even a small change in the protein’s structure will change the enzyme’s shape enough to prevent the substrate from binding, and thus keep the reaction from occurring. In this laboratory, you will demonstrate how altering the temperature, enzyme concentration, and pH will affect an enzyme’s ability to function. Specifically, you will be studying the actions of the enzyme catalase.

Exercise 1: Catalase Activity

Materials:

• Water (distilled or deionized)
• Disposable pipets
• Catalase (400 units/mL solution)
• Hydrogen peroxide (3% solution)
• Sucrose solution (5% solution)
• Small test tubes (10 ml test tubes) for Exercises 1, 2, and 4
• Large test tubes (20 ml test tubes) for Exercise 3
• Ruler
• Wax pencil / Sharpie
• Ice
• Boiling water bath
• Refrigerator
• Incubator (37o Celsius)
• 0.1 M HCl
• 0.1 M NaOH
• Buffer pH 7.0
• Safety glasses

Catalase is an enzyme that speeds up the breakdown of hydrogen peroxide to water and oxygen:

Hydrogen peroxide → water + oxygen gas

\[\ce{2 H2O2 → 2 H2O + O2}\]

• What is the reactant in this reaction? _______________________________

• What is the substrate for catalase? ________________________________

• What are the products for this reaction? _____________________________

• Bubbling occurs as the reaction proceeds. Explain why. _____________________________________________________________

Procedure:

1. With a wax pencil (or Sharpie), label three small test tubes (#1, #2, #3).
2. Using the ruler, mark each tube at the 1 cm (first mark) and 5 cm (second mark) levels from the bottom of the tube.
3. Fill tube #1 to the first mark with catalase. Then fill it to the second mark with hydrogen peroxide.
4. Swirl the contents to mix and wait at least 20 seconds for bubbles to develop.
5. Using the ruler, measure the height of the bubble column (in millimeters), and record your results in Table 1 below.
6. Fill tube #2 to the first mark with water. Then fill it to the second mark with hydrogen peroxide.
7. Swirl the contents to mix and wait at least 20 seconds for bubbles to develop.
8. Using the ruler, measure the height of the bubble column (in millimeters), and record your results in Table 1 below.
9. Fill tube #3 to the first mark with catalase. Then fill to the second mark with sucrose solution.
10. Swirl the contents to mix and wait at least 20 seconds for bubbles to develop.
11. Using the ruler, measure the height of the bubble column (in millimeters), and record your results in Table 1 below.

Table 1: Catalase Activity

Tube	Contents	Bubble Column Height (mm)
1	catalase, hydrogen peroxide
2	water, hydrogen peroxide
3	catalase, sucrose solution

• Which tube showed the most bubbling? Explain why. ________________________________________________________________

________________________________________________________________

• Which tube was the negative control? __________________________________

• Which tube was the positive control? __________________________________

• Describe what you observe in test tube #3. What was the purpose of this tube? ________________________________________________________________

________________________________________________________________

Exercise 2: Effect of Temperature on Enzyme Activity

In general, cold temperatures slow chemical reactions, and warm temperatures speed up chemical reactions. Every enzyme, however, has an optimal temperature at which it works best. Some enzymes prefer cooler temperatures, and others prefer warm temperatures. However, extreme increases in temperature (i.e. boiling) will denature enzymes, making them inactive.

In the following exercise, you will test enzyme function at four different temperatures (on ice, in a refrigerator, in a warm incubator, and in a boiling water bath). Before setting up this experiment, formulate a hypothesis regarding the effect on enzyme activity. For example, do you think the enzyme will perform better at a warmer temperature? A cooler temperature? On ice? At boiling temperatures?

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. With a wax pencil (or Sharpie), label four small test tubes (#1, #2, #3, #4) and mark each at the 1 cm and 5 cm levels.

2. Fill each tube to the first mark with catalase.

3. Place tube #1 on ice, tube #2 at room temperature, tube #3 in a 37 degree Celsius incubator or warm water bath, and tube #4 in a boiling water bath or heat block. Wait 15 minutes.

4. While you are waiting, use a thermometer to measure the temperature of the environment for all four tubes. Record the values in Table 2 below.

5. After the 15-minute incubation, fill each tube to the second mark with hydrogen peroxide.

6. Swirl the contents to mix and wait 20 seconds.

7. Measure the height of the bubble column (in mm) for each tube and record your results in Table 2 below.

Table 2: Effect of Temperature on Enzyme Activity

Tube	Temperature (oC)	Bubble Column Height (mm)
1 - on ice
2 - room temperature
3 - warm water bath
4 - boiling

• Which tube showed the most enzyme activity? Explain. ________________________________________________________________

• What is the optimal temperature for catalase activity? Explain ________________________________________________________________

• Was your hypothesis supported? ________________________________________________________________

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

• What is the optimal temperature for enzymes in the human body? ________________________________________________________________

• What effect could a fever have on enzymatic activity in the human body? ________________________________________________________________

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

1. Graph your results using the data from Table 2.

2. What is the dependent variable? Which axis is used to graph this data? ______________________________________________________________________

3. What is your independent variable? Which axis is used to graph this data? ______________________________________________________________________

Exercise 3: Effect of Concentration on Enzyme Activity

In general, the amount of product produced in a given amount of time should increase if you increase the enzyme concentration.

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________
3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. With a wax pencil (or Sharpie), label three large test tubes (#1, #2, #3). Make sure to use the larger tubes provided by your instructor.

2. Mark tube #1 at the 1 cm and 5 cm levels.

3. Fill to the first mark with catalase and to the second mark with hydrogen peroxide.

4. Swirl the contents to mix and wait 10 seconds.

5. Measure the height of the bubble column (in mm) and record your results in Table 3 below.

6. Mark tube #2 at the 2 cm and 6 cm levels.

7. Fill to the first mark with catalase and to the second mark with hydrogen peroxide.

8. Swirl the contents to mix and wait 10 seconds.

9. Measure the height of the bubble column (in mm) and record your results in Table 3 below.

10. Mark tube #3 at the 3 cm and 7 cm levels.

11. Fill to the first mark with catalase and to the second mark with hydrogen peroxide.

12. Swirl the contents to mix and wait 10 seconds.

13. Measure the height of the bubble column (in mm) and record your results in Table 3.

Table 3: Effect of Concentration on Enzyme Activity

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

• The amount of bubbling corresponds to the degree of enzyme activity. Which tube showed the most activity. Explain. ________________________________________________________________

________________________________________________________________

• If we waited for an unlimited amount of time, would the results be the same in all tubes? Explain. ________________________________________________________________

________________________________________________________________

• In this experiment, was the amount of substrate the same in all three tubes? ________________________________________________________________

• Generate a hypothesis about what you might observe if the amount of substrate concentration were increased while the enzyme concentration remained the same. Explain. ________________________________________________________________

________________________________________________________________

Extension Activity: (Optional)

The results of this experiment can be presented graphically. The presentation of your data in a graph will assist you in interpreting your results. Based on your results, you can complete the final step of scientific investigation, in which you must be able to propose a logical argument that either allows you to support or reject your initial hypothesis.

1. Graph your results using the data from Table 3.

2. What is the dependent variable? Which axis is used to graph this data? ______________________________________________________________________

3. What is your independent variable? Which axis is used to graph this data? ______________________________________________________________________

Exercise 4: Effect of pH on Enzyme Activity

Each enzyme has an optimal pH, or level of acidity or alkalinity at which it functions best. A higher or lower pH affects hydrogen bonding and can alter the structure of the enzyme, leading to reduced activity.

Employing Steps in the Scientific Method:

1. Record the Question that is being investigated in this experiment. ________________________________________________________________

2. Record a Hypothesis for the question stated above. ________________________________________________________________

3. Predict the results of the experiment based on your hypothesis (if/then). ________________________________________________________________

4. Perform the experiment below and collect your data.

Procedure:

1. With a wax pencil (or Sharpie), label three small test tubes (#1, #2, #3) and mark at the 1 cm, 2 cm, and 6 cm levels. Fill each tube to the 1 cm level with catalase.

2. Fill tube #1 to the second mark with 0.1 M HCl.

3. Fill tube #2 to the second mark with buffer (pH 7.0).

4. Fill tube #3 to the second mark with 0.1 M NaOH.

5. Carefully swirl to mix and wait 20 seconds.
6. Fill all three tubes to the third mark with hydrogen peroxide.

7. Carefully swirl to mix and wait 20 seconds.

8. Measure the height of the bubble column (in mm) for each tube and record your results in Table 4 below.

Table 4: Effect of pH on Enzyme Function

Tube	Acid / Buffer / Base	Bubble Column Height (in mm)
1	0.1 M HCl
2	Buffer pH 7.0
3	0.1 M NaOH

• Which tube showed the most activity? ________________________________________________________________

• What is the optimal pH for catalase? ________________________________________________________________

Questions for Review

1. What is the function of an enzyme? What specific monomers (i.e. building blocks) make up enzymes?
2. What does it mean when an enzyme becomes denatured?

Describe at least 2 ways in which an enzyme can become denatured.

3. What types of bonds or interactions are broken when an enzyme becomes denatured (name at least 3)?

4. Explain the difference between substrate and active site.

5. If an enzyme functions well at a pH of 4, would you expect it to also work well at a pH of 7? Explain.

Practical Challenge

1. Eggs can contain bacteria such as Salmonella. Considering what you’ve learned in this laboratory exercise, explain how cooking eggs makes them safe to eat.
2. What can happen to an enzyme when the pH of the environment becomes too acidic or too alkaline? Use your observations from the lab to support your answer.
3. If you don’t want a cut potato to get brown, what could you do to prevent it assuming that the ‘browning’ occurs because of an enzymatic reaction? Explain your answer.
4. What would happen to enzyme activity if the pH of your blood decreased (became more acidic) or increased (became more basic)? Explain.

References

Belwood, Jacqueline; Rogers, Brandy; and Christian, Jason, Foundations of Biology Lab Manual (Georgia Highlands College). “Lab 5: Enzyme Function,” (2019). Biological Sciences Open Textbooks. 18. CC-BY https://oer.galileo.usg.edu/biology-textbooks/18