APBiology Unit 1, Chapter 5 Research Question How do abiotic or biotic factors influence the rates of enzymatic reactions? Background Enzymes are the catalysts of biological systems. They speed up chemical reactions in biological systems by lowering the activation energy, the energy needed for molecules to begin reacting with each other. Enzymes do this by forming an enzyme-substrate complex that reduces energy required for the specific reaction to occur. Enzymes have specific shapes and structures that determine their functions. The enzyme s active site is very selective, allowing only certain substances to bind. If the shape of an enzyme is changed in any way, or the protein denatured, then the binding site also changes, thus disrupting enzymatic functions. Enzymes are fundamental to the survival of any living system and are organized into a number of groups depending on their specific activities. Two common groups are catabolic enzymes ( cata or kata- from the Greek to break down ) for instance, amylase breaks complex starches into simple sugars and anabolic enzymes ( a- or an- from the Greek to build up ). (You may know this second word already from stories about athletes who have been caught using anabolic steroids to build muscle.) Catalytic enzymes, called proteases, break down proteins and are found in many organisms; one example is bromelain, which comes from pineapple and can break down gelatin. Bromelain often is an ingredient in commercial meat marinades. Papain is an enzyme that comes from papaya and is used in some teeth whiteners to break down the bacterial film on teeth. People who are lactose intolerant cannot digest milk sugar (lactose); however, they can take supplements containing lactase, the enzyme they are missing. All of these enzymes hydrolyze large, complex molecules into their simpler components; bromelain and papain break proteins down to amino acids, while lactase breaks lactose down to simpler sugars. Anabolic enzymes are equally vital to all living systems. One example is ATP synthase, the enzyme that stores cellular energy in ATP by combining ADP and phosphate. Another example is rubisco, an enzyme involved in the anabolic reactions of AP Lab 13 Enzyme APUnit 1 2012 Page 1 of 11
building sugar molecules in the Calvin cycle of photosynthesis. To begin this investigation, you will focus on the enzyme peroxidase obtained from a turnip, one of numerous sources of this enzyme. Peroxidase is one of several enzymes that break down peroxide, a toxic metabolic waste product of aerobic respiration. Using peroxidase, you will develop essential skills to examine your own questions about enzyme function. Later, you will have an opportunity to select an enzyme, research its properties and mode of reaction, and then design an experiment to explore its function. The investigation also provides an opportunity for you to apply and review concepts you have studied previously, including the levels of protein structure, energy transfer, abiotic and biotic influences on molecular structure, entropy and enthalpy, and the role of enzymes in maintaining homeostasis. In an enzyme-catalyzed reaction, the substance to be acted upon (the substrate = S) binds reversibly to the active site of the enzyme (E). One result of this temporary union is a reduction in the energy required to activate the reaction of the substrate molecule so that the products (P) of the reaction are formed. In summary: E + S ES E + P Note that the enzyme is not changed in the reaction and can be recycled to break down additional substrate molecules. Each enzyme is specific for a particular reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure. The active site is the portion of the enzyme that interacts with the substrate, so that any substance that blocks or changes the shape of the active site affects the activity of the enzyme. A description of several ways enzyme action may be affected follows. 1. Salt concentration. If the salt concentration is close to zero, the charged amino acid side chains of the enzyme molecules will attract each other. The enzyme will denature and form an inactive precipitate. If, on the other hand, the salt concentration is very high, normal interaction of charged groups will be blocked, new interactions will occur, and again the enzyme will precipitate. An intermediate salt concentration such as that of human blood (0.9%) or cytoplasm is the optimum for many enzymes. 2. ph. ph is a logarithmic scale that measures the acidity or H+ concentration in a solution. The scale runs from 0 to 14 with 0 being highest in acidity and 14 lowest. When the ph is in the range of 0-7, a solution is said to be acidic; if the ph is around 7, the solution is neutral; and if the ph is in the range of 7-14, the solution is basic. Amino acid side chains contain groups such as COOH and NH 2 that readily gain or lose H+ ions. As the ph is lowered an enzyme will tend to gain H+ ions, and eventually enough side chains will be affected so that the enzyme s shape is disrupted. Likewise, as the ph is raised, the enzyme will lose H+ ions and eventually lose its active shape. Many enzymes perform optimally in the neutral ph range and are denatured at either an extremely high or low ph. Some enzymes, such as pepsin, which acts in the human stomach where the ph is very low, have a low ph optimum. AP Lab 13 Enzyme APUnit 1 2012 Page 2 of 11
3. Temperature. Generally, chemical reactions speed up as the temperature is raised. As the temperature increases, more of the reacting molecules have enough kinetic energy to undergo the reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster with increasing temperature. However, if the temperature of an enzyme-catalyzed reaction is raised still further, a temperature optimum is reached; above this value the kinetic energy of the enzyme and water molecules is so great that the conformation of the enzyme molecules is disrupted. The positive effect of speeding up the reaction is now more than offset by the negative effect of changing the conformation of more and more enzyme molecules. Many proteins are denatured by temperatures around 40-50 C, but some are still active at 70-80 C, and a few even withstand boiling. 4. Activations and Inhibitors. Many molecules other than the substrate may interact with an enzyme. If such a molecule increases the rate of the reaction it is an activator, and if it decreases the reaction rate it is an inhibitor. These molecules can regulate how fast the enzyme acts. Any substance that tends to unfold the enzyme, such as an organic solvent or detergent, will act as an inhibitor. Some inhibitors act by reducing the -S-Sbridges that stabilize the enzyme s structure. Many inhibitors act by reacting with side chains in or near the active site to change its shape or block it. Many well-known poisons such as potassium cyanide and curare are enzyme inhibitors that interfere with the active site of critical enzymes. The enzyme used in this lab, catalase (a peroxidase), has four polypeptide chains, each composed of more than 500 amino acids. This enzyme is ubiquitous in aerobic organisms. One function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes. Catalase might also take part in some of the many oxidation reactions that occur in all cells. The primary reaction catalyzed by catalase is the decomposition of H 2 0 2 to form water and oxygen. 2 H 2 O 2 2 H 2 O +O 2 (gas) In the absence of catalase, this reaction occurs spontaneously, but very slowly. Catalase speeds up the reaction considerably. In this experiment, a rate for this reaction will be determined. Much can be learned about enzymes by studying the kinetics (particularly the changes in rate) of enzyme-catalyzed reactions. For example, it is possible to measure the amount of product formed, or the amount of substrate used, from the moment the reactants are brought together until the reaction has stopped. Figure 3.1: Enzyme Activity If the amount of product formed is measured at regular intervals and this quantity is plotted on a graph, a curve like the one that follows is obtained. Study the solid line on the graph of this reaction. At time 0 there is no product. After 30 seconds, 5 micromoles ( mo1es) have been formed; after one minute, 10 moles; after two minutes, 20 moles. The AP Lab 13 Enzyme APUnit 1 2012 Page 3 of 11
rate of this reaction could be given as 10 moles of product formed per minute for this initial period. Note, however, that by the third and fourth minutes, only about 5 additional moles of product have been formed. During the first three minutes, the rate is constant. From the third minute through the eighth minute, the rate is changing; it is slowing down. For each successive minute after the first three 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. In the comparison of the kinetics of one reaction with another, a common reference point is needed. For example, suppose you wanted to compare the effectiveness of catalase obtained from potato with that of catalase obtained from liver. It is best to compare the reactions when the rates are constant. In the first few minutes of an enzymatic reaction such as this, the number of substrate molecules is usually so large compared with 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 nearly constant rate. The slope of the graph line during this early period is called the initial rate of the reaction. The initial rate of any enzymecatalyzed reaction is determined by the characteristics of the enzyme molecule. It is always the same for any enzyme and its substrate at a given temperature and ph. This also assumes that the substrate is present in excess. The rate of the reaction is the slope of the linear portion of the curve. To determine a rate, pick any two points on the straight-line portion of the curve. Divide the difference in the amount of product formed between these two points by the difference in time between them. The result will be the rate of the reaction which, if properly calculated, can be expressed as moles product/sec. The rate then is: Or from the graph, In the graph above, the rate between 2 and 3 minutes is calculated: The rate of a chemical reaction may be studied in a number of ways, including the following: 1. Measuring the rate of disappearance of substrate (in this example, H 2 O 2 ); 2. Measuring the rate of appearance of product (in this case, O 2, which is given off as a gas); 3. Measuring the heat released (or absorbed) during the reaction. AP Lab 13 Enzyme APUnit 1 2012 Page 4 of 11
In this laboratory you will observe the conversion of hydrogen peroxide (H 2 0 2 ) to water and oxygen gas by the enzyme catalase. You will then measure the amount of oxygen generated and calculate the rate of the enzyme-catalized reaction. Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities of living organisms. They act as catalysts, substances that speed up chemical reactions without being destroyed or altered during the process. Enzymes are extremely efficient and may be used over and over again. One enzyme may catalyze thousands of reactions every second. Both the temperature and the ph at which enzymes function are extremely important. Most organisms have a preferred temperature range in which they survive, and their enzymes most likely function best within that temperature range. If the environment of the enzyme is too acidic or too basic, the enzyme may irreversibly denature, or unravel, until it no longer has the shape necessary for proper functioning. H 2 O 2 is toxic to most living organisms. Many organisms are capable of enzymatically destroying the H 2 O 2 before it can do much damage. H 2 O 2 can be converted to oxygen and water, as follows: 2 H 2 O 2 2O + O 2 Although this reaction occurs spontaneously, enzymes increase the rate considerably. At least two different enzymes are known to catalyze this reaction: catalase, found in animals and protists, and peroxidase, found in plants. A great deal can be learned about enzymes by studying the rates of enzyme-catalyzed reactions. The rate of a chemical reaction may be studied in a number of ways including: measuring the rate of appearance of a product (in this case, O 2, which is given off as a gas) measuring the rate of disappearance of substrate (in this case, H 2 O 2 ) measuring the pressure of the product as it appears (in this case, O 2 ). In this experiment, you will measure the rate of enzyme activity under various conditions, such as different enzyme concentrations, ph values, and temperatures. It is possible to measure the concentration of oxygen gas formed as H 2 O 2 is destroyed using an O 2 Gas Sensor. If a plot is made, it may appear similar to the graph shown. At the start of the reaction, there is no product, and the concentration is the same as the atmosphere. After a short time, oxygen accumulates at a rather constant rate. The slope of the curve at this initial time is constant and is called the initial rate. As the peroxide is destroyed, less of it is available to react and the O 2 is produced at lower rates. When no more peroxide is left, O 2 is no longer produced. AP Lab 13 Enzyme APUnit 1 2012 Page 5 of 11
Learning Objectives To understand the relationship between enzyme structure and function To make some generalizations about enzymes by studying just one enzyme in particular To determine which factors can change the rate of an enzyme reaction To determine which factors that affect enzyme activity could be biologically Important Key Vocabulary Baseline is a universal term for most chemical reactions. In this investigation the term is used to establish a standard for a reaction. Thus, when manipulating components of a reaction (in this case, substrate or enzyme), you have a reference to help understand what occurred in the reaction. The baseline may vary with different scenarios pertinent to the design of the experiment, such as altering the environment in which the reaction occurs. In this scenario, different conditions can be compared, and the effects of changing an environmental variable (e.g., ph) can be determined. Rate can have more than one applicable definition because this lab has two major options of approach, i.e., using a color palette and/or a spectrophotometer to measure percent of light absorbance. When using a color palette to compare the change in a reaction, you can infer increase, decrease, or no change in the rate; this inference is usually called the relative rate of the reaction. When using a spectrophotometer (or other measuring devices) to measure the actual percent change in light absorbance, the rate is usually referred to as absolute rate of the reaction. In this case, a specific amount of time can be measured, such as 0.083 absorbance/minute. Before doing this laboratory 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; and that changes in temperature, ph, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions. After doing this laboratory you should be able to: measure the effects of changes in temperature, ph, enzyme concentration, and substrate concentration on reaction rates of an enzyme-catalyzed reaction in a controlled experiment; and explain how environmental factors affect the rate of enzyme-catalyzed reactions. MATERIALS computer Vernier computer interface Logger Pro 600 ml beaker enzyme suspension four 18 X 150 mm test tubes AP Lab 13 Enzyme APUnit 1 2012 Page 6 of 11
Vernier Gas Pressure Sensor ice 1-hole rubber stopper assembly ph buffers 10 ml graduated cylinder test tube rack 250 ml beaker of water thermometer 3% H 2 O 2 three dropper pipettes Vernier O 2 Sensor Biochamber for O 2 sensor PROCEDURE 1. Obtain and wear goggles. 2. Connect the Gas Pressure Sensor to the computer interface. Prepare the computer for data collection by opening the file 02 (Pressure) Enzyme from the Advanced Biology with Vernier folder of Logger Pro. 3. Connect the plastic tubing to the valve on the Gas Pressure Sensor. 1 2 3 4 Figure 1 Part I Testing the Effect of Enzyme Concentration 4. Place four test tubes in a rack and label them 1, 2, 3, and 4. Partially fill a beaker with tap water for use in Step 5. 5. Add 3 ml of water and 3 ml of 3% H 2 O 2 to each test tube. 6. Using a clean dropper pipette, add 1 drop of enzyme suspension to Test Tube 1. Note: Be sure not to let the enzyme fall against the side of the test tube. Table 1 Test tube label Volume of 3% H 2 O 2 (ml) Volume of water (ml) 1 3 3 2 3 3 3 3 3 4 3 3 AP Lab 13 Enzyme APUnit 1 2012 Page 7 of 11
7. Stopper the test tube and gently swirl to thoroughly mix the contents. The reaction should begin. The next step should be completed as rapidly as possible. 8. Connect the free-end of the plastic tubing to the connector in the rubber stopper as shown in Figure 2. Click to begin data collection. Data collection will end after 3 minutes. 9. If the pressure exceeds 130 kpa, the pressure inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130 kpa. 10. When data collection has finished, disconnect the plastic tubing connector from the rubber stopper. Remove the rubber stopper from the test tube and discard the contents in a waste beaker. 11. Find the rate of enzyme activity: a. Move the mouse pointer to the point where the data values begin to increase. Hold down the mouse button. Drag the mouse pointer to the point where the pressure values no longer increase and release the mouse button. b. Click the Linear Fit button,, to perform a linear regression. A floating box will appear with the formula for a best-fit line. c. Record the slope of the line, m, as the rate of enzyme activity in Table 4. d. Close the linear regression floating box. 12. Find the rate of enzyme activity for test tubes 2 4: a. Add 2 drops of the enzyme solution to test tube 2. Repeat Steps 7 11. b. Add 3 drops of the enzyme solution to test tube 3. Repeat Steps 7 11. c. Add 4 drops of the enzyme solution to test tube 4. Repeat Steps 7 11. Part II Testing the Effect of Temperature 13. Place four clean test tubes in a rack and label them T 0 5, T 20 25, T 30 35, and T 50 55. 14. Add 3 ml of 3% H 2 O 2 and 3 ml of water to each test tube, as shown in Table 2. Table 2 Test tube label Volume of 3% H 2 O 2 (ml) Volume of water T 0 5 3 3 T 20 25 (room temp) 3 3 T 30 35 3 3 T 50 55 3 3 Figure 2 AP Lab 13 Enzyme APUnit 1 2012 Page 8 of 11
15. Measure the enzyme activity at 0 5 C: a. Prepare a water bath at a temperature in the range of 0 5 C by placing ice and water in a 600 ml beaker. Check that the temperature remains in this range throughout this test. b. Place Test Tube T 0 5 in the cold water bath until the temperature of the mixture reaches a temperature in the 0 5 C range. Record the actual temperature of the test-tube contents in the blank in Table 4. c. Add 2 drops of the enzyme solution to Test Tube T 0 5. Repeat Steps 7 11. 16. Measure the enzyme activity at 30 35 C: a. Prepare a water bath at a temperature in the range of 30 35 C by placing warm water in a 600 ml beaker. Check that the temperature remains in this range throughout this test. b. Place Test Tube T 30 35 in the warm water bath until the temperature of the mixture reaches a temperature in the 30 35 C range. Record the actual temperature of the test-tube contents in the blank in Table 4. c. Add 2 drops of the enzyme solution to Test Tube T 30 35. Repeat Steps 7 11. 17. Measure the enzyme activity at 50 55 C: a. Prepare a water bath at a temperature in the range of 50 55 C by placing hot water in a 600 ml beaker (hot tap water will probably work fine). Check that the temperature remains in this range throughout this test. b. Place Test Tube T 50 55 in the warm water bath until the temperature of the mixture reaches a temperature in the 50 55 C range. Record the actual temperature of the test-tube contents in the blank in Table 4. c. Add 2 drops of the enzyme solution to Test Tube T 50 55. Repeat Steps 7 11. 18. Measure the enzyme activity at 20 25 C (room temperature): a. Record the temperature of Test Tube T 20 25 in Table 4. b. In the tube labeled T 20 25, add 2 drops of the enzyme solution. Repeat Steps 7 11. Part III Testing the Effect of ph 19. Place three clean test tubes in a rack and label them ph 4, ph 7, and ph 10. 20. Add 3 ml of 3% H 2 O 2 and 3 ml of each ph buffer to each test tube, as in Table 3. Table 3 ph of buffer Volume of 3% H 2 O 2 (ml) Volume of buffer (ml) ph 4 3 3 ph 7 3 3 ph 10 3 3 21. In the tube labeled ph 4, add 2 drops of the enzyme solution. Repeat Steps 7 11. 22. In the tube labeled ph 7, add 2 drops of the enzyme solution. Repeat Steps 7 11. AP Lab 13 Enzyme APUnit 1 2012 Page 9 of 11
23. In the tube labeled ph 10, add 2 drops of the enzyme solution. Repeat Steps 7 11. DATA 1 Drop 2 Drops 3 Drops 4 Drops Test tube label 0 5 C range: C 20 25 C range: C 30 35 C range: C 50 55 C range: C ph 4 ph 7 ph 10 Table 4 Slope, or rate (kpa/min) PROCESSING THE DATA Enzyme concentration plot 1. On Page 2 of this experiment file, create a graph of the rate of enzyme activity vs. enzyme concentration. The rate values should be plotted on the y-axis, and the number of drops of enzyme on the x-axis. The rate values are the same as the slope values in Table 4. Temperature plot 2. On Page 3 of this experiment file, create a graph of the rate of enzyme activity vs. temperature. The rate values should be plotted on the y-axis, and the temperature on the x-axis. The rate values are the same as the slope values in Table 4. ph plot 3. On Page 4 of this experiment file, create a graph of rate of enzyme activity vs. ph. The rate values should be plotted on the y-axis, and the ph on the x-axis. The rate values are the same as the slope values in Table 4. QUESTIONS Part I Effect of Enzyme Concentration 1. How does changing the concentration of enzyme affect the rate of decomposition of H 2 O 2? AP Lab 13 Enzyme APUnit 1 2012 Page 10 of 11
2. What do you think will happen to the rate of reaction if the concentration of enzyme is increased to five drops? Predict what the rate would be for 5 drops. Part II Effect of Temperature 3. At what temperature is the rate of enzyme activity; Highest?, Lowest? Explain. 4. How does changing the temperature affect the rate of enzyme activity? Does this follow a pattern you anticipated? 5. Why might the enzyme activity decrease at very high temperatures? Part III Effect of ph 6. At what ph is the rate of enzyme activity;h Highest? Lowest? 7. How does changing the ph affect the rate of enzyme activity? AP Lab 13 Enzyme APUnit 1 2012 Page 11 of 11