ENZYMES: BIOLOGICAL CATALYSTS OF LIFE

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1 Potential Energy Lab 6 ENZYMES: BIOLOGICAL CATALYSTS OF LIFE OBJECTIVES Define catalyst, enzyme, activation energy, enzyme-substrate complex, substrate, product, active site, denaturation, and cofactor; Explain how enzymes operate; Recognize benzoquinone as a brown substance formed in damaged plant tissue; Indicate the substrates for the enzyme catechol oxidase; Describe the effect of temperature on the rate of chemical reactions in general and on enzymatically controlled reactions in particular; Describe the effect that an atypical ph may have on enzyme action; Indicate how a cofactor might operate and identify a cofactor for catechol oxidase. INTRODUCTION Of all the elements and compounds that help make life possible, none ranks higher in importance than the group of molecules we call enzymes. Enzymes are proteins that function as biological or organic catalysts. A catalyst is a substance that lowers the amount of energy necessary for a chemical reaction to proceed. You might think of this so-called activation energy as a hump to be negotiated. Enzymes decrease the size of the hump, in effect turning a mountain into a molehill (Figure 6-1). Substr ate Activation energy with enzyme Activation energy w/ out enzyme Energy released as product forms Reaction Progress Figure 6-1 Enzymes and activation energy. 1

2 By lowering the activation energy, an enzyme affects the rate at which reaction occurs. Enzyme- boosted reactions may proceed from 0 thousand to million times faster than they would without the enzyme. Most enzymes are protein molecules, and each enzyme has at least one cleft or groove known as the active site, where the substrate (reactant or the substance being acted upon) attaches and forms a temporary enzyme-substrate complex. Products are formed, and the enzyme molecule is released unchanged. Thus, the enzyme is not used up in the process and is capable of catalyzing the same reaction repeatedly. This process is summarized below: Substrate + Enzyme Enzyme-Substrate Complex Products + Enzyme Although thousands of enzymes are present within cells, we will examine only one, catechol oxidase (also known as tyrosinase), to demonstrate the effects of several factors that influence enzyme action. These factors include: 1. Temperature; 2. ph (hydrogen ion concentration of the environment); 3. Specificity (how discriminating the enzyme is in catalyzing different substrates); 4. Cofactor necessity (the need for a metallic ion for enzyme activity). I. FORMATION AND DETECTION OF BENZOQUINONE This exercise will investigate the result of catechol oxidase activity. In the presence of oxygen, catechol oxidase catalyzes the removal of electrons and hydrogens from catechol, a phenolic compound found in plant cells. Catechol is converted to benzoquinone, a pigment product. The hydrogens combine with oxygen, forming water (Figure 6-2). The pigment products are responsible for the darkening of fruits and vegetables, such as apples and potatoes, after exposure to air. H O H C O H C C H C C C H H + 1 / 2 O 2 O H C H C C + C C H C H O H 2 O Catech ol (substra te) Figure 6-2 The oxidation of catechol. Benzoquinone (product) In this experiment, you will use a potato extract to test for the presence of catechol oxidase and to establish the appearance of the product (brown) when the reaction takes place. CAUTION: Some of the chemicals (Catechol, Hydroquinone) used in these experiments may be hazardous to your health if they are ingested or taken in through your skin. Wash your hands after each experiment. 2

3 Procedure 1. With a grease pencil, label three test tubes I 1, I 2, and I 3. Place your initials on each test tube for later identification. 2. Lay the test tubes against a metric ruler and mark lines on the tubes corresponding to 1 cm and 2 cm from the bottom of each tube. 3. Fill each tube as follows: Potato Extract Catechol Distilled Water I 1 1 cm 1 cm I 2 1 cm 1 cm I 3 1 cm 1 cm 4. Shake all test tubes using your finger to flick the bottom of the tube or a vortex mixer. 5. NOTE: RETURN POTATO EXTRACT TO ICE BATH IMMEDIATELY AFTER USE. 6. Record the color of the solution in each tube in the Time 0 spaces of Table 6-1 Table 6-1 Formation and Detection of Benzoquinone Time (minutes) 0 I 1 [Potato Extract + Catechol] I 2 [Potato Extract + dh 2 O] I 3 [Catechol + dh 2 O] 7. Place the tubes in the 40 C water bath. 8. Remove the tubes after min and record the color of the solution of each tube in Table Remove the tubes from the water bath and save them for comparison of results of other experiments you will perform. After minutes, the catechol should be completely oxidized. The color of the product in tube I 1 will be considered to be a "5" on a color intensity scale of 0 to 5, while the color of the substance in tubes I 2 and I 3 will be considered to be "0". You will use this scale to make comparisons in experiments II to V. Save these tubes for comparison with tubes from other exercises. These are your controls. Fill in Table 6-2. Table 6-2 Color Intensity Scale Intensity No. Color 0 I 2 and I 3 5 I 1 3

4 Questions 1. What is the brown-colored substance that appeared in test tube I 1? 2. What was the substrate for the reaction that occurred in tube I 1? 3. What was the product of the reaction in tube I 1? 4. What substances lacking in tubes I 2 and I 3 account for the absence of the brown-colored substance? 5. What is the purpose of having tubes I 2 and I 3? II. ENZYME SPECIFICITY Generally, enzymes are substrate-specific, acting on one particular substrate or a small number of structurally similar substrates. This specificity is due to the three-dimensional structure of the enzyme. In order for the enzyme-substrate complex to form, the structure of the substrate must complement very closely that of the active site of the enzyme. The active site is a special region of the enzyme to which the substrate binds. Although the active site has a small amount of moldability, you can think of and enzyme as a key and the substrate as the particular lock into which it fits, as illustrated in Figure 6-3. Substrate Enzyme-substrate complex Products Active site 1. Substrate molecule approaches active site. 2. Substrate molecule binds to active site; reaction takes place. 3. Product molecules leave active site; enzyme is unaltered. Figure 6-3 Induced-fit model of enzyme-substrate interactions. This experiment demonstrates the ability of the enzyme catechol oxidase to catalyze the oxidation of two different but structurally similar substrates: catechol and hydroquinone. 4

5 Examine the chemical structure of each compound in Figure 6-4. OH OH OH OH Catechol Hydroquinone Figure 6-4 You need not memorize these structural formulas, but do notice that both are ring structures with two hydroxyl ( OH) groups attached. Keep this in mind as you do the next experiment, in which you will determine how specific (discriminating) catechol oxidase is for particular substrates. Procedure 1. With a grease pencil, label two CLEAN test tubes II 1, and II 2. Include your initials for identification. 2. Lay the test tubes against a metric ruler and mark lines indicating 1 cm and 2 cm from the bottom of each tube. 3. Fill each tube as follows: Potato Extract Catechol Hydroquinone II 1 1 cm 1 cm II 2 1 cm 1 cm 4. Shake all test tubes using a vortex mixer. 5. NOTE: RETURN POTATO EXTRACT TO ICE BATH IMMEDIATELY AFTER USE. 6. Compare the color intensity of the solution in each test tube with the standards produced in Experiment I and record at Time 0 in Table Place the test tubes in a 40 C water bath. 8. Examine the test tubes after minutes, recording the color intensity (scale 0 to 5) of the contents of each in Table

6 Table 6-3 Specificity of Catechol Oxidase Relative Color Intensity on a Scale of 0 to 5 Time (minutes ) 0 II 1 [Potato Extract + Catechol] II 2 [Potato Extract + Hydroquinone] 8. After completing this experiment, use the test tube brush provided to wash test tubes. Thoroughly wash them with soap, rinse with tap water and place them in the test tube rack so they may drain properly. Finally, tidy up your work area making certain all materials and equipment are available for the next class. Questions 1. Upon which substrate does catechol oxidase work best, forming the most benzoquinone in the shortest amount of time? 2. Based upon your knowledge of the structure of the two substrates (in Figure 6-4) what apparently determines the specificity of catechol oxidase? III. EFFECT OF TEMPERATURE ON ENZYME ACTIVITY The rate at which chemical reactions take place is largely determined by the temperature of the environment. Generally, for every C rise in temperature, the reaction rate doubles. Within a rather narrow range, this is true for enzymatic reactions also. However, because enzymes are proteins, excessive temperature alters their structure, destroying their ability to function. When an enzyme's structure is changed sufficiently to destroy its function, the enzyme is said to be denatured. Most enzymatically-controlled reactions have an optimum temperature and ph, that is, one temperature and ph where activity is maximized. Procedure 1. Half fill one 400 ml beaker with tap water. Add a few boiling chips and turn on the hotplate to the highest temperature setting. Bring the water to a boil, and then turn the heat down so that the water just continues to boil. 2. Put 150 ml of tap water into a second beaker, and then add ice to the water. 3. Half fill a third beaker with water from the source at room temperature. 4. With a grease pencil, label six test tubes III 1 through III 6. Include your initials for identification. 5. Lay the test tubes against a metric ruler and mark off lines indicating 1 cm and 2 cm from the bottom of each tube. 6. Fill each tube as to the 1-cm mark with Potato Extract containing Catechol Oxidase. 6

7 7. NOTE: RETURN POTATO EXTRACT TO ICE BATH IMMEDIATELY AFTER USE. 7

8 8 a. Place tube III 1 in the 400 ml beaker of ice water. Measure the H 2 O temperature and record here: C. b. Place tube III 2 in the 400 ml beaker containing room temperature water. Room temperature is: C. c. Place tube III 3 in the 40 C water bath. d. Place tube III 4 in the 60 C water bath. e. Place tube III 5 in the 80 C water bath. f. Place tube III 6 in the 400 ml beaker containing boiling water. The boiling water is at: C 9. Allow the test tubes to remain at the various temperatures for 5 minutes.. Remove the tubes and add 1% Catechol to the 2-cm line on each. Agitate the tubes using the vortex mixer to mix the contents. CAUTION: Wear a Heat-Resistant Glove When Handling Heated Glassware. 11. Record in Table 6-4 the relative color intensity (scale 0 to 5) of the solution in each tube, using the standard established in Experiment I. Return each tube to its respective temperature bath immediately after recording. Table 6-4 Effect of Temperature on Enzyme Action Relative Color Intensity on a Scale of 0 to 5 Time (minutes) III 1 C III 2 C III 3 40 C III 4 60 C III 5 80 C III 6 C Plot the data from Table 6-4 for the -minute reading on the graph in Figure

9 Figure 6-5. Effect of temperature on enzymes activity. 14. When you are done, use the test tube brush provided to wash test tubes. Thoroughly wash them with soap, rinse with deionized water and place them in the test tube rack so they may drain. Finally, tidy up your work area making certain all equipment and materials are there for the next class. Questions 1. Over what temperature range does catechol oxidase remain active? 2. What is the optimum (best) temperature for activity of this enzyme? 3. What happens to enzymes exposed to very high temperatures? IV. EFFECT OF ph ON ENZYME ACTIVITY Another factor influencing the rate of enzyme catalysis is the hydrogen ion concentration (ph) of the solution. ph, like temperature, affects the three-dimensional shape of enzymes, thus regulating their function. Most enzymes operate best when the ph of the solution is near neutrality (ph 7). Others, however, have ph optima in the acidic or basic range, corresponding to the environment in which they normally are found. This experiment will allow you to determine the ph optimum of catechol oxidase. Procedure 1. With a grease pencil, label seven test tubes IV 1 through IV 7. Include your initials for identification. 2. Lay the test tubes against a metric ruler and mark lines indicating 4 cm, 5 cm and 6 cm from the bottom of each tube. 9

10 3. Take your test tubes to the location of the phosphate buffer series and fill each tube according to the following directions: ph Buffer Potato Extract 1% Catechol IV 1 ph 2 (4 cm) 1 cm 1 cm IV 2 ph 4 (4 cm) 1 cm 1 cm IV 3 ph 6 (4 cm) 1 cm 1 cm IV 4 ph 7 (4 cm) 1 cm 1 cm IV 5 ph 8 (4 cm) 1 cm 1 cm IV 6 ph (4 cm) 1 cm 1 cm IV 7 ph 12 (4 cm) 1 cm 1 cm 4. Add catechol oxidase to each tube and mix. Incubate for 5 min then add catechol. 5. Agitate the tubes by using a vortex mixer. 6. Record in Table 6-5 at Time 0 the relative color intensity of each tube immediately after adding the 1% Catechol. Table 6-5 Effect of ph on Enzyme Activity Relative Color Intensity on a Scale of 0 to 5 Time (min) IV 1 ph 2 IV 2 ph 4 IV 3 ph 6 IV 4 ph 7 IV 5 ph 8 IV 6 ph IV 7 ph Place the tubes in the 40 C water bath. 8. Agitate the tubes frequently over the next minutes. Record in Table 6-5 the relative color intensity of each tube. 9. Plot the data from Table 6-5 for your -minute reading on the graph in Figure 6-6. Be sure to label your x and y axis properly.

11 Figure 6-6. Effect of ph on enzyme activity.. When you are done, use the test tube brush provided to wash test tubes. Thoroughly wash them with soap, rinse with tap water and place them in the test tube rack so they may drain. Finally, tidy up your work area making certain all equipment and materials are there for the next class. Questions 1. What is the range of ph over which catechol oxidase catalyzes catechol to benzoquinone? 2. What is the optimum ph for activity of catechol oxidase? V. NECESSITY OF COFACTORS FOR ENZYME ACTIVITY Some enzymatic reactions occur only when the proper cofactors are present. Cofactors are metallic ions that are part of the structure of the active site, making possible the formation of the enzyme-substrate complex. Catechol oxidase contains copper. In this experiment, we will use Phenylthiourea (PTU), which binds strongly to copper, to remove copper ions. Thus, we will be able to determine if copper is a cofactor necessary for producing benzoquinone from catechol. Procedure 1. With a grease pencil, label two test tubes V 1 and V 2. Include your initials for identification. 2. Lay the test tubes against a metric ruler and mark lines indicating 1 cm and 2 cm from the bottom of each tube. 3. NOTE: RETURN POTATO EXTRACT TO ICE BATH IMMEDIATELY AFTER USE. 11

12 Potato Extract * PTU 1% Catechol V 1 1 cm few crystals 1 cm V 2 1 cm 1 cm * Use a spatula or scoop for this procedure. 4. Agitate the contents of the tubes with the vortex mixer. Record at Time 0 in Table 6-6 the relative color intensities (scale 0 to 5). 5. Place the tubes in a 40 C water bath. Agitate the tubes several times during the next minutes. 6. Remove the tubes from the water bath and compare their relative color intensities. Record your observations in Table 6-6. Table 6-6 Is Copper a Cofactor for Catechol Oxidase? Relative Color Intensity on a Scale of 0 to 5 Time (minutes ) 0 V 1 [Potato Extract + PTU] V 2 [Potato Extract + no PTU] 7. When you are done, use the test tube brush provided to wash test tubes. Thoroughly wash them with soap, rinse with tap water and place them in the test tube rack so they may drain. Finally, tidy up your work area making certain all equipment and materials are there for the next class. Questions 1. Did Benzoquinone form in tube V 1? 2. Did Benzoquinone form in tube V 2? 3. From this experiment, what can you conclude about whether or not copper is needed for catechol oxidase activity? 4. What substance used in this experiment contained copper? 12

13 POST-LAB QUESTIONS 1. Eggs can contain bacteria such as Salmonella. Considering what you ve learned in this exercise, explain how cooking eggs makes them safe to eat. 2. As you demonstrated in this experiment, high temperatures inactivate catechol oxidase. How is it that some bacteria live in the hot springs of Yellowstone Park at temperatures as high as 73 C? 3. Why do you think high fevers alter cellular functions? 4. Some surgical procedures involve lowering a patient s body temperature during periods when blood flow must be restricted. What effect might this have on enzymecontrolled cellular metabolism? 5. At one time, it was believed that individuals who had been submerged under water for longer than several minutes could not be resuscitated. Recently this has been shown to be false, especially if the person was in cold water. Explain why cold-water drowning victims might survive prolonged periods under water. 6. Explain what happens to catechol oxidase when the ph is on either side of the optimum. 7. What would you expect the ph optimum to be for an enzyme secreted into your stomach? 8. Is it necessary for a cell to produce one enzyme molecule for every substrate molecule that needs to be catalyzed? Why or why not? 9. Explain the difference between substrate and active site.. Describe a few strategies one could use to prevent apples from browning after slicing them. 13