A. Learning Objectives
In this lab, students will:
• analyze the effect of catechol oxidase on the production of benzoquinone.
• design and conduct experiments to study how physical conditions affect enzyme activity.
• plot data graphically.
B. Textbook Correlation
Please review Section 6.2 Enzymes and Ribozymes in Chapter 6: An Introduction to Energy, Enzymes, and Metabolism to assist in writing the intrroduction and researching the experiment.
C. Introduction
Please write a two paragraph introduction to enzymes;
Paragraph #1: Discuss the structure/function of enzymes. In your discssion, address the make-up of most enzymes, the role of the active site and its impact on specificity, and the idea behind the induced fit theory, Also discuss activation energy and how enzymes speed up chemical reactions by impacting the activation energy.
| INSERT IMAGE OF TYPICAL ENZYME | INSERT AN IMAGE OF AN ENERGY DIAGRAM WITH ACTIVATION ENERGY LABELED. THIS IMAGE SHOULD ALSO DEMONSTRATE HOW ENZYMES IMPACT ACTIVATION ENERGY. | INSERT AN IMAGE OF THE CATALYTIC CYCLE OF AN ENZYME |
| figure legend | FIGURE LEGEND. | FIGURE LEGEND. |
Paragraph #2: Discuss how enzyme activity is regulated in a cell. Include in the discussion the idea of enzyme saturation, how saturation is overcome, the physical requirements for optimal enzyme activity, and the role of inhibitors (both competitive and non-competitive). When discussing inhibitors, include the idea behind allosteric regulation.
| INSERT AN IMAGE SHOWING A PLOT OF ENZYME ACTIVITY VS SUBSTRATE CONCENTRATION. THIS IMAGE SHOULD DEMONSTRATE ENZYME SATURATION BY SUSBTRATES. | INSERT AN IMAGE OR ANIMATION DEMONSTRATING THE ROLES OF COMPETITIVE VS NON-COMPETITIVE INHIBTORS | INSERT AN IMAGE/ANIMATION SHOWING ALLOSTERIC REGULATION OF ENZYMES WITHIN A CELL |
| FIGURE LEGEND | FIGURE LEGEND | FIGURE LEGEND |
In today’s exercise you will first observe the actions of the enzyme catechol oxidase. After this exercise you will be ready to design two experiments on your own to test the physical requirements for optimal enzyme activity.
D. Catechol Oxidase Activity
In today’s exercise the enzyme you will use is catechol oxidase. In plants this copper-containing enzyme creates brown pigment when exposed to air (specifically oxygen), and it is the reason fruits turn brown after they are sliced. The brown color is due to the production of the product benzoquinone, a substance that is toxic to food-spoiling bacteria. When the peel is damaged, oxygen can then react with the catechol, protecting the fruit.
In this experiment, we will test catechol oxidase activity. The enzyme is extracted from potatoes using a blender and is referred to as potato extract in the subsequent experiments.
Experimental Procedure:
1. Label 3 test tubes 1–3.
2. Pipette the amount of catechol and water into the appropriate test tube as outlined in Table 1. Do not add the catechol oxidase to all tubes until just before starting the incubation in step 3.
|
Tube |
mL of Catechol |
mL of Water |
mL of Catechol Oxidase |
|
1 |
1 |
0 |
1 mL (20 drops) |
|
2 |
0 |
1 |
1 mL (20 drops) |
|
3 |
1 |
1 |
0 mL (0 drops) |
3. Place the test tubes in the 37⁰C water bath for 10 minutes.
4. Record your results in the table above. Use the following scale:
0 no color change
1 little color change
2 more color change
3 dark color change
|
Tube |
Result |
Conclusion |
|
1 |
|
|
|
2 |
|
|
|
3 |
|
|
Questions
1. Which tube is the negative control? Which tube is your positive control?
2. What would it mean if tube 2 turned brown?
E. Design an Experiment to Study Enzyme Activity Under Different Physical Conditions.
Protein activity is highly dependent on its three-dimensional structure. Conditions that cause a protein to denature (unfold) results in the loss of protein activity. Environmental deviations from optimal cause an enzyme to lose activity. Question: What is the optimal temperature for catechol oxidase activity? What is the ideal pH for catechol oxidase activity? What is the optimal salinity for catechol oxidase activity? Use the experiment from section D as a template. Remember to include positive and negative controls when applicable. Make sure you take photographic images of your results and a video of your procedure explaining how you designed the experiment.
Materials Provided:
Experiment #1: Temperature
1. Hypothesis:
2. Experimental Design:
Experiment #2: pH
1. Hypothesis:
2. Experimental design:
Experiment #3: Salt
1. Hypothesis:
2. Experimental design:
Please embed your presentation below.