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.
Enzymes are catalysts, which means that they increase the rate of chemical reactions without themselves being changed or consumed. They increase the speed of the reaction by lowering the amount of activation energy needed for the reaction to proceed. This is done by either straining the bonds of reactants in the reaction so that they may more easily achieve the transition state, or positioning the reactants close together so that they can form bonds. Also, the enzyme may temporarily change the local environment so that the activation energy for the reaction is lower. An important feature to note on the enzyme is the active site, which is where the chemical reaction takes place (where the substrate binds). The site is very specific to the substrate that the enzyme binds with, so enzymes usually only bind with one type of substrate. This is known as the “lock and key” theory, because only the right substrate (key) will fit into the active site (lock). The induced fit theory states that, when a substrate binds with its enzyme, the enzyme will go through conformational changes in order to better fit the substrate. This means that the “lock and key” theory is not completely correct, since a lock does not change shape in order to better fit its key.
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The figure shows an enzyme and its active site, along with the substrate that the enzyme binds to.
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The diagram depicts an exergonic reaction, in which energy is lost. The activation energy needed for the reaction to proceed is shown as lower with the enzyme catalyst than without the enzyme catalyst. |
The figure shows the catalytic cycle of an enzyme. First, the substrate binds with the active site. The enzyme then goes through conformational changes in order to better accommodate the the substrate. The activation energy is then lowered so that the reaction can be carried out, and the products are released. |
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.
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This graph shows the rate of the reaction carried out by an enzyme as substrate concentration increases. Note that the horizontal asymptote represents the maximum speed of the reaction.
Note that the graph begins to plateau after substrate concentration reaches a certain point. This shows how the reaction rate cannot become any faster after substrate concentration is high enough. This is an example of saturation.
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The yellow figures are enzymes. The active site is the site in which the blue figure would go. The allosteric site is the site in which the green figure would goThe purple figures are substrates. The blue figure is a competitive inhibitor and the green figure is a noncompetitive inhibitor.
Note that the competitive inhibitor binds to the active site while the noncompetitive inhibitor binds to the allosteric site.
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The green figures are enzymes. The notches that the orange and red figures are getting binded to/repelled from are the active sites. The notches that the yellow and purple molecules are binding to are the allosteric sites. The orange and red figures represent substrates. The yellow and purple figures are enhancer molecules or noncompetitive inhibitors.
Note that binding of molecules to the allosteric site could either enable or inhibit enzyme function.
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Enzyme activity is regulated in a few different ways. Enzymes often use reversible inhibitors to change how enzymes function, and thus regulate them. Competitive inhibitors are molecules that bind noncovalently to the active site of an enzyme and inhibit the ability of the substrate to bind. Competitive inhibitors compete with the binding ability of the substrate that the enzyme would usually bind to. They do this by mimicking the structure of the substrate. Competitive inhibitors literally block the active site, so that substrates cannot bind to the active site. Competitive inhibitors increase K_m for the substrate, meaning that a higher concentration for substrate is needed to achieve the same rate of the chemical reaction. The second type of reversible inhibitor is noncompetitive inhibitors. Noncompetitive inhibitors bind noncovalently to an enzyme at a location outside the active site, called an allosteric site, and inhibits the enzyme’s function. It’s also important to recognize how binding of molecules to allosteric sites affects enzyme function. When a molecule binds to the allosteric site of the enzyme, the enzyme undergoes a conformational change. This changes the orientation/structure of the active site of the enzyme, possibly inhibiting it from binding substrates (it should also be noted that binding of molecules to allosteric sites could activate enzymes as well). These types of inhibitors are reversible because the types of bonds that they make are noncovalent, meaning that they can be reversed.
Concentration of the substrate can also affect enzyme activity. Generally, the more substrate there is in an enzyme’s environment, the faster the enzyme will perform its reactions. However after a certain point of substrate concentration, all of the active sites on the enzyme will be filled. This is called saturation. The rate of the reaction will begin to plateau and slow down once the enzyme is saturated. Saturation can be overcome with the bonding of a molecule to the allosteric site, possibly increasing the rate of the reaction despite saturation of active sites due to a conformational change in the enzyme.
However if the environment of an enzyme does not have the right conditions, the enzyme will not function to begin with. Two of the most important environmental conditions that affect enzyme function are temperature and pH level. Enzymes have a specific range of temperature and pH level that they function optimally at. For example, if temperature is just a few degrees above what is optimal for an enzyme, the function of the enzyme is inhibited greatly. If the temperature is too high, the enzyme might even begin to unfold and lose its three-dimensional shape, a process called denaturing. As mentioned before, enzymes also require specifc pH levels for them to function optimally. Even if the pH of the environment of an enzyme is only slightly different from the optimal pH, enzyme function begins to be inhibited.
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
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mL of Catechol
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mL of Water
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mL of Catechol Oxidase
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1
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1
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0
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1 mL (20 drops)
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2
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0
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1
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1 mL (20 drops)
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3
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1
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1
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0 mL (0 drops)
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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
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Result
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Conclusion
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1
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2
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3
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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: We predict that Catechol Oxidase will work best at ~21 degrees Celsius, which is considered room temperature. This is because room temperature is the approximate condition under which catechol oxidase would normally have to work.
2. Experimental Design:
- Begin by filling four test tubes with ~1 mL of potato extract in each one, and label them 1-4.
- Obtain and create three water baths, one of 20 degrees Celsius, another of 30 degrees Celsius, and a third of 40 degrees Celsius.
- Place test tubes 1-3 in the 20, 30, and 40 degree water baths respectively and let them rest for five minutes.
- Fill all four test tubes with 1 mL of catechol. Test tube 4 will be the control, as there will be no change in enzyme activity without a change in temperature. Test tube 4 should be left in a normal environment and not placed in a bath.
- Let the test tubes sit in their appropriate baths/environments for five minutes.
- Remove the test tubes and record the color change, specifically noting the darkness of the tube.
Experiment #2: pH
1. Hypothesis: Catechol Oxidase will work best at a more neutral pH of about 7.
2. Experimental design:
- Label 7 test tubes with the numbers 1-7, and fill them with the pH buffers of 2, 4, 6, 7, 8, 10, and 12 respectively.
- The tube with the pH of 7 will be the control, as we know that to be the neutral pH, so enzyme activity should not be affected
- To each tube, add 1 mL of catechol and 1 mL of potato extract.
- Agitate/stir the tubes.
- Let the tubes sit for five minutes and then record the color change, in the same vein as experiment one.
Experiment #3: Salt
1. Hypothesis: Catechol Oxidase will work best at a salinity of 0%, as that is the preferred salinity of the organisms that use this enzyme.
2. Experimental design:
- We are going to create five test tubes with the differing salinity including: 0%, 2.5%, 5%, 7.5%, and 10%.
- To create those solutions, add the following amounts of water and 10% stock NaCl solution:
- 20 mL of water, 0 mL of NaCl (0% salinity)
- 15 mL of water, 5 mL of NaCl (2.5% salinity)
- 10 mL of water, 10 mL of NaCl (5% salinity)
- 5 mL of water, 15 mL of NaCL (7.5% salinity)
- 0 mL of water, 20 mL of NaCl (10% salinity)
- One control will be the 0% salinity solution, as we should expect no change in normal enzyme activity. The other control will be the 10% salinity solution, as very few, if any, enzymes can work in such salient conditions.
- Add 1 mL of catechol and 1 mL of potato extract to each test tube.
- Let each tube sit for five minutes and then record the results with the same parameters as the previous two experiments.
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