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 discussion, 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. 

               Enzymes are a class of catalysts. Catalysts are substances that speed up the rate of a chemical reaction without being consumed or permanently affected in the reaction. Enzymes are proteins and they are the most common types of catalysts in living cells. Reactions that are catalyzed with enzymes are millions of times faster than catalyzed reactions. Enzymes act in living cells by lowering the activation energy of reactions. When the reactants of a reaction try to come closer, there is a repulsion between them. This repulsion can only be overcome with an initial burst of energy, activation. However, this activation energy is prevents products from being formed rapidly. Enzymes function in lowering this activation energy, in order for the rapid production of products to occur. After lowering the activation energy enzymes also help the reactants reach a transition state, which is when original bonds in the reactants are stretched to their limit. This state must be reached in order for the reaction to continues. Enzymes strain reactants and bring reactants closer together, making it easier for the reactants to achieve a transition state. 

     Enzymes are large proteins that bind to small reactants, called substrates. By binding to reactants, enzymes assist the reactants reach a transition state. Substrates bind to enzymes at their active site, which is the location in the enzymes where the chemical reaction will occur. Enzymes are very specific to which substrates they will bind to. When the enzyme and substrate are bound the resulting structure is called an enzyme-substrate complex. The enzyme-substrate complex exhibit a phenomenon called induced fit; this is when enzymes undergo confromational changes in order to perfectly fit the substrates that they are binding to. Enzymes also have allosertic sites. Noncompetitive inhibitors bind to the allosteric sites in order to stop enzymes activity. This is a cycle; substrates bind to enzymes at the active sites and produce products. Noncompetitive Inhibitors attach to the the active site of the molecules and stop the function of the enzymes. When the product is needed again, the substrate binds to the enymes at the active sites. 

	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.

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.

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

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:

• test tubes
• plastic pipettes
• catechol
• potato extract
• water baths (3) and hot plate
• ice
• thermometers 
• phosphate buffers ranging from pH of 2 to 12 (actual buffers available (in pH units): 2, 4, 6, 7, 8, 10, 12) 
• distilled water 
• 10% NaCl stock solution

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.