ENZYME CATALYSIS OVERVIEW In this laboratory you will use a chemical titration to measure and then calculate the rate of conversion of hydrogen peroxide (H2O2) to water and oxygen using the enzyme catalase. OBJECTIVES Before doing this exercise, you should understand: the general functions and activities of enzymes the relationship between the structure and function of enzymes the concept of initial reaction rates of enzymes how the concept of free energy relates to enzyme activity that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions. After doing this exercise, you should be able to: measure the effects of changes of temperature, pH, enzyme concentration, and substrate concentration on rates of an enzyme-catalyzed reaction in a controlled experiment. explain how environmental factors affect the rte of enzyme-catalyzed reactions. INTRODUCTION Enzymes are proteins produced by living cells. They act as catalysts in biochemical reactions. A catalyst speeds up the rate of a chemical reaction and makes it possible for the reaction of occur with a lower initial input of energy. One benefit of enzyme catalysis is that the cell can carry out complex chemical activities at a relatively low temperature. In biochemical reactions the enzyme, E, combines reversibly with its specific substrate, S, to form a complex, ES. One result of this temporary union is a reduction in the energy required to activate the reaction of the substrate molecule so the P, the products of the reaction, are formed. In summary: E + S → ES → E + P Note that the enzyme is not changed in the reaction and can break down additional substrate molecules. Each enzyme is specific for a particular reaction. In practice, this specificity means that if you mix a purified substrate with crude preparations of enzyme that might contain many other substances, the reaction you are interested in will still only be catalyzed by that one enzyme. This means you can do a quantitative assay of the amount of enzyme present, even if the enzyme preparation is not completely pure. The enzyme you will be working with is catalase. Catalase has a molecular weight approximately 240, 000 daltons and contains 4 polypeptide chains, each composed of more than 500 amino acid residues. This enzyme occurs universally in aerobic organisms. One function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide (H2O2) formed as a by-product of metabolic processes. Catalase might also take part in some of the many oxidation reactions going on in all cells. The primary reaction catalyzed by catalase is the decomposition of H 2O2 to form water and oxygen: 2 H2O2 → 2 H2O + O2 (gas) This reaction occurs spontaneously, but not at a very rapid rate. Catalase speeds up the reaction considerably. In this experiment, a rate for this reaction will be determined. If you measure the amount of product formed (or substrate used) from the moment reactants are brought together in a chemical reaction until the reaction stops, you will observe that the reaction rate changes over the life of the reaction. Consider the following example. The amount of product formed is measured at 30 second intervals and this quantity is plotted on a graph, giving the curve in Figure 1. Observe the solid line for this reaction. At time 0 there is no product. After 30 seconds, 5 μmoles have been formed; after 1 minute, 10 μmoles; after 2 minutes, 20 μmoles. The rate of this reaction could be given as 10 μmoles of product formed per minute for this initial period, Note, however, that by the third and fourth minutes, only about 5 additional μmoles of product have been formed. During the first 3 minutes, the rate is constant. From the third minute through the eighth minute, the rate is decreasing. For each successive minute after the first 3 minutes, the amount of product formed in that interval is less than in the preceding minute. From the seventh minute onward, the reaction rate is very slow. 2 Figure 1 40 Product (μ moles) 30 20 10 1 2 3 4 5 6 7 8 Time (minutes) To understand this observation, you need to keep in mind that essentially two things must happen for the reaction products to form: the enzyme must form the complex with its substrate (E + S → ES ) and then the reaction must occur (ES → E + P ). Once the enzyme-substrate complex is formed, the reaction will occur at the same rate as long as temperature and pH are constant. To form the complex, the enzyme and substrate molecules must collide in the right orientation to fit together. The more total collisions between enzyme and substrate that happen per unit time, the more chance of a successful collision in that time interval.. You can probably visualize that the number of collisions per unit time would be affected by the concentration of substrate and enzyme. Therefore the rate of formation of the ES complex is affected by the concentration of substrate and enzyme in the reaction mixture. In the first few minutes of an enzymatic reaction such as this, the number of substrate molecules is usually so large compared to the number of enzyme molecules that changing the substrate concentration does not (for a short period at least) affect the number of successful collisions between substrate and enzyme. During this early period, the enzyme is acting on substrate molecules at a constant rate. The slope of the graph line during this early period is called the initial velocity of the reaction. The initial velocity (or rate) of any enzyme-catalyzed reaction is determined by the characteristics of the enzyme molecule. It is always the same for an enzyme and it substrate as long as temperature and pH are constant and the substrate is present in excess. The rate of the reaction, therefore, is the slope of the linear portion of the curve. The slope of the linear portion in Figure 1 is 0. 17 μmoles/second. PART 1: TESTING FOR CATALASE ACTIVITY 1. To observe the reaction to be studied, use the labeled syringe to transfer 10 ml of 1.5 % (0.44 M H 2O2) into your unlabeled 60 ml plastic cup. Use a plastic transfer pipet(1 ml syringe) to add 1 ml of the catalase solution. The bubbles coming from the reaction mixture are the O 2 resulting from the breakdown of H2O2 by catalase. 2. To demonstrate the effect of boiling on enzymatic activity, use the same transfer pipet to transfer 5 ml of purified catalase extract to a test tube. Set the transfer pipet aside. Place the test tube containing catalase in a boiling water bath to 5 minutes. While the catalase boils, rinse out the unlabeled plastic cup you used in step 1 for reuse. Transfer 10 ml of 1.5 % H2O2 into the unlabeled 60 ml plastic cup. Use a fresh plastic transfer pipet (1 ml syringe) to add 1 ml of the boiled catalase solution. 3. To demonstrate the presence of catalase in living tissue, cut 1 cm3 of potato and liver and macerate (smush with vigor) them. Rinse your unlabeled 60 ml plastic cup again, and add 10 ml fresh 1.5 % H 2O2. Add the potato to the cup and observe. Repeat with the liver. GENERAL NOTES ON PROCEDURE FOR PARTS 2 – 4. The rate of a chemical reaction may be studied in a number of ways, including the following: 1. 2. 3. Measuring the rate of disappearance of substrate; in this example H 2O2. Measuring the rate of appearance of product; in this case O 2 which is given off as a gas. Measuring the heat released (or used) during the reaction. 3 In this experiment the disappearance of the substrate, H2O2, is measured as follows: 1. A purified catalase extract is mixed with substrate, H 2O2, in a beaker. The enzyme catalyzes the conversion of H2O2 to H2O and O2 (gas). 2. Before all of the H2O2 is converted to H2O and O2 the reaction is stopped by adding sulfuric acid H2SO4). The H2SO4 lowers the pH, denaturing the enzyme and therefore stopping the enzyme’s catalytic activity. 3. After the reaction is stopped, the amount of substrate (H 2O2) remaining in the beaker is measured. To assay this quantity, potassium permanganate is used. Potassium permanganate (KMnO 4), in the presence of H2O2 and H2SO4, reacts as follows: 5 H2O2 + 2 KMnO4 + 3 H2SO4 → H2SO4 + 2 MnSO4 + 8 H2O + 5 O2 H2O2 is essential for this reaction. Once all the H2O2 has reacted, additional KMnO4 will be in excess and will not be decomposed. The addition of excess KMnO4 causes the solution to have a permanent pink or brown color. Therefore, the amount of H2O2 remaining is determined by adding KMnO4 until the KMNO4 color no longer disappears and the whole mixture stays a faint pink or brown, permanently. Add no KMnO 4 after this point. The amount of KMnO4 added is a proportional measure of the amount of H 2O2 remaining ( 1 molecule of KMnO4 decomposes 2.5 molecules of H2O2). THE GENERAL PROCEDURE O2 H2SO4 Catalase H2O2 Catalase H2O2 H2SO4 Catalase H2O2 Measuring of Remaining H2O2 GENERAL NOTES OF PROCEDURE This assay involves performing a titration with KMnO4. You will be using a syringe for the procedure. When using a syringe, first draw a little air into the syringe, then, draw in the KMnO4 solution. The initial volume reading is here the meniscus of the solution (no the syringe plunger) intersects the scale on the syringe. In order to get good results in all these experiments, you must not cross-contaminate syringes and beakers. Be sure to read the directions carefully and use the appropriately labeled equipment. Take time to be sure you select the correct instrument before you use it. For example, substituting the H 2SO4 syringe to the H2O2 syringe could affect your results. PART 2: DETERMINING THE BASELINE To determine the amount of H2O2 initially present in your nominally 1.5 % solution, you need to perform all the steps of the procedure without adding catalase (enzyme) to the reaction mixture. This amount is known as the baseline and is an index of the initial concentration of H 2O2 in solution. In any series of experiments, a baseline should be established first. 4 You must wear goggles during this procedure!!! Use extreme care handling acids!!!!! Goggles!!! 1. Using the 10 – 12 ml syringe labeled H2O2. put 10 ml of 1.5 % H2O2 in the 60 ml plastic cup/beaker labeled Baseline. 2. Use the plastic transfer pipet (1ml syringe) to all 1 ml of distilled water (instead of enzyme solution) fro the distilled water cup/beaker. 3. Using the 10 – 12 ml syringe labeled H2SO4. add 10 ml of 1.0 M H2SO4 from the labeled beaker. 4. Mix well. 5. Using the 5 ml syringe labels Transfer, remove 5 ml of the reaction mixture and put it in the cup labeled Titration and assay for the amount of H2O2 present. Rinse the Transfer syringe by drawing up some water from the Wash Water cup. Handle KMnO4 with care. Avid contact with skin and eyes! Goggles!!! If you are going to use a syringe to do the titration, draw about 0.2 ml air, then about 5 ml of KMnO4 solution and record the initial volume reading. Use the syringe to add KMnO 4 one drop at a time to the solution in the Titration cup until a persistent pink or brown color is obtained. Gently swirl the solution after adding each drop. When you get the persistent pink or brown color, record the final volume of KMnO4 in the Baseline Data Table. Subtract the final reading from the initial reading to determine the amount of KMnO4 used. Remember, the amount of KMnO4 used is proportional to the amount of H 2O2 that was in the solution. PART 3: AN ENZYME – CATALYZED RATE OF H2O2 DECOMPOSITION In this experiment, you will determine the rate at which a 1.5% H 2O2 solution decomposes when the reaction is catalyzed by catalase. To do this, you should determine how much H 2O2 has been consumed after 10, 30, 60, 120, and 180 seconds. If a day or more has passed since you determined the baseline by finding the amount of H 2O2 present in your 1.5 % solution, you will have to redo part 1 to get a baseline. PROCEDURE Set up the 60 ml plastic cups with the labels 10 sec, 30 sec, 60 sec, 120 sec, and 180 sec. Use the 10 – 12 ml syringe labeled H2O2 to add 10 mls H2O2 to each cup. Do one time point assay at a time. One student should add reagents and a different student should keep time. Read the directions thoroughly before beginning. For the 10 sec and 30 sec tests, you will need to have the H 2SO4 measured and ready before beginning the assay. Remember to wear goggles!!!!! Use extreme care handling acids!!!!! Handle KMnO4 with care. Avid contact with skin and eyes! Goggles!!! 10 seconds 1. Add 1 ml of catalase extract. 2. Swirl gently for 10 seconds. 3. At 10 seconds, add 10 ml of H2SO4 (1.0 M). 5 30 seconds 1. Add 1 ml of catalase extract. 2. Swirl gently for 30 seconds. 3. At 30 seconds, add 10 ml of H2SO4 (1.0 M). 60, 120, and 180 seconds Each time, repeat steps 1 through 3, as above, except allow the reactions to proceed for 60, 120, and 180 seconds, respectively. TITRATIONS Use the rinsed Transfer syringe to remove 5 ml from the 10 sec reaction cup and place it in the plastic cup labeled Titration. Rinse the transfer syringe by drawing in water from the Wash Water cup and expelling it into the Waste Cup. Assay for the amount of H2O2 remaining in the reaction cup by titrating with KMnO4. As you did in Part 2 in the Baseline determination, use a syringe to add KMnO4 a drop at a time until the solution remains pink or brown. Gently swirl the Titration Cup after each addition. Should the end point be overshot, there is sufficient sample left to repeat the titration. Record your results in the Enzyme – Catalyzed Reaction Data Table. PART 4: THE UNCATALYZED RATE OF H2O2 DECOMPOSITION Left alone, H2O2 spontaneously decomposes to water and oxygen at a slow rate. Therefore, a solution that contained 1.5 % H2O2 at the time of preparation does not necessarily contain 1.5 % H 2O2 after storage. In addition, the preparation many not have been perfectly precise. PROCEDURE Remember to wear goggles!!!!! Use extreme care handling acids!!!!! Handle KMnO4 with care. Avid contact with skin and eyes! Goggles!!! 1. To determine the rate of spontaneous conversion of H 2O2 to H2O and O2 in an uncatalyzed reaction, transfer approximately 15 ml of 1.5 % H2O2 to the 60 ml cup labeled H2O2 Overnight. Store it uncovered at room temperature for approximately 24 hours. 2. Use the syringe labeled H2O2 to transfer 10 mls of H2O2 from the H2O2 Overnight cup (from step 1) to the 60 ml plastic cup labeled Uncatalyzed Decomposition. 3. Use the plastic transfer pipet (1 ml syringe) to add 1 ml of distilled water (instead of enzyme solution) from the distilled water cup. 4. Using the 10 – 12 ml syringe labeled H2SO4, add 10 ml of 1.0 M H2SO4 from the labeled beaker. 5. Mix well. 6. Using the 5 ml syringe labeled Transfer, remove 5 ml of the reaction mixture and put it in the cup labeled Titration and assay for the amount of H2O2 present. Rinse the Transfer syringe by drawing up some water from the Wash water cup and expelling it into the Waste cup. 7. Titrate with KMnO4 as did in Parts 2 and 3. 8. Record your data in the Uncatalyzed Rate of H 2O2 Decomposition Data Table.