Impact of Enzyme, Substrate, pH, Ion, Temp, and Inhibitor on Catalase Reaction Rates - Pro, Study Guides, Projects, Research of Physiology

Download Impact of Enzyme, Substrate, pH, Ion, Temp, and Inhibitor on Catalase Reaction Rates - Pro and more Study Guides, Projects, Research Physiology in PDF only on Docsity! THE EFFECTS OF CHANGES IN ENZYME CONCENTRATION, SUBSTRATE CONCENTRATION, pH, ION CONCENTRATION, TEMPERATURE AND PRESENCE OF INHIBITOR ON REACTION RATES OF AN ENZYME CATALYZED REACTION Hannah Matos, Hannah Greer, Nicole Palminteri, Sierra Kokoszka Course: Biology 270 (Human Physiology) § 5 Instructor: Dr. Norman Garrison James Madison University Department of Biology September 24, 2008 Introduction Enzymes are catalysts that bind temporarily to one or more of the reactants of the reaction they catalyze. In doing so, they lower the activation energy needed to create a reaction (Enzymes, 2004). These reactions are necessary to control a living system’s activity. With the presence of enzymes, the majority of cellular reactions occur about a million times faster than they would without the enzyme (Ophardt, 2003). Most enzymes only react with one substrate, or reactant, to produce products. An example of one of these reactants is catalase which speeds up the decomposition of water, breaking it down into basic hydrogen and oxygen. This experiment focused on enzyme inhibitors and the effect that different concentrations of a given inhibitor has on the reaction rate. Inhibitors are materials that slow, or stop in some cases, the rate of the chemical reaction. Each type of enzyme has a specific optimal pH and temperature at which they are able to catalyze reactions more efficiently. The human body caters to these specific needs by controlling the necessary pH levels and temperatures (Enzymes, 2004). There are three categories of inhibitors. A competitive inhibitor occurs when a substrate and a substance resembling the substrate are added to the enzyme. The lock and key theory is used to explain how this type of inhibitor works. All enzymes have a specific shape and the given substrate that reacts with the enzyme fits into the mold of the enzyme. When a competitive inhibitor is introduced, it resembles the shape of the substrate and competes to fit within the mold of the enzyme. The more inhibitor present, the more difficult it becomes for the substrate to react with the enzyme, therefore slowing the reaction. Recent enzyme studies at the U.S. Department of Energy’s Brookhaven National Laboratory have looked at adenoviruses and the enzymes that trigger their effects. These specific enzymes, called protease, have three different binding sites and researchers are Steps 1-5 were repeated using three catalase-soaked disks and concentrations of NaCl in the substrate solution. For the three trials, 1.5% of H2O2 solution containing 0% NaCl, 2% NaCl, and 10% NaCl were used, respectively. D. INFLUENCE OF TEMPERATURE ON CATALASE ACTIVITY Steps 1-5 were repeated using three catalase-soaked disks and 10 mL of 3% H2O2 in different temperature water baths. For each trial, the temperature of the reaction vessel was stabilized for 2-3 minutes in the water bath before a run was made. In the first through fifth trials, un-boiled catalase were used for water-bath temperatures at 5°C, 21°C, 33°C, 37°C, and 41.1°C, respectively. In the last trial, “boiled” catalase extract (100°C) was used in a room temperature water bath. E. INFLUENCE OF ENZYME INHIBITOR ON CATALASE ACTIVITY Steps 1-5 were repeated using three disks soaked with catalase at different concentration of copper sulfate and 10 mL of 3% H2O2. For trials 1-5, three drops of 0.1 M, 0.25 M, 0.5 M, 0.75 M, and 1.0 M CuSO4 solution were added into 2.5 mL catalase solution, respectively. Each tube was mixed in a vortex machine then the mixture was poured into a weigh boat and sat for five minutes before testing the enzyme activity. Results The catalase concentration on the total catalase activity was directly proportional to the reaction velocity (Figure 1). An increase in catalase concentration (3 soaked catalase disks) steadily increased the rate of reaction. As the substrate concentration increased, the reaction velocity increased until it reached the maximum at 10% H2O2 at 124.8 ± 57 mL of O2/min (Figure 2). After this point, increases in substrate concentration did not increase the velocity. As pH increased, the reaction velocity increased until the optimum pH of 7 was reached. pH greater than 7 led to a decline from 96.9 ± 11 mL of O2/min to 87.1 ± 18 mL of O2/min (Figure 3). An increase in NaCl concentration on catalase activity steadily decreased the reaction velocity (Figure 4). The increase in temperature led to an increase in activity until the optimum temperature of 41°C was reached. After this point, the velocity collapsed to 0 mL of O2/min at 100°C (Figure 5). The velocity dropped with an increase in concentrations of enzyme inhibitor, CuSO4 (Figure 6). 0 20 40 60 80 100 120 0 1 2 3 4 Number of Disks V e lo c it y ( m L O 2/ m in ) Figure 1. Influence of catalase concentration on total activity. 0 20 40 60 80 100 120 140 0 2 4 6 8 10 12 H2O2 Concentration (%) V e lo c it y ( m L O 2/ m in ) Figure 2. Influence of substrate concentration on catalase activity. 0 20 40 60 80 100 120 0 2 4 6 8 10 12 pH value V e lo c it y ( m L o f O 2/ m in ) Figure 3. Influence of pH on catalase activity. 0 10 20 30 40 50 0 2 4 6 8 10 12 NaCl concentration (%) V e lo c it y ( m L o f O 2/ m in ) Figure 4. Influence of ionic concentration on catalase activity. and ionic bonds. If the shape is altered, the enzyme ceases to function as the active site and no longer accommodates the substrate. The enzyme becomes denatured. Inhibitors are substances which alter the catalytic action of the enzyme by competing with the substrate for the active sites (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow or stop the rate of reaction. An excess of substrates blocks the active site so that the enzyme goes unused and prevents any other substrate molecules from occupying them causing the reaction rate to drop. Conclusions 1. Three catalase soaked disks had a greater influence on enzyme catalase activity than one catalase soaked disk because an increase in enzyme concentration increases the amount of O2 produced. 2. The enzyme catalase reaction rate increased with increasing concentrations of the substrate. At higher substrate concentrations the rate began to level off. Once the maximum reaction rate was achieved, further increases in substrate concentration had no effect. 3. At extreme levels of acidity and basicity (less than or greater than the optimum pH of 7), the enzyme molecule resulted in a change in conformation that decreased the enzyme activity. 4. Enzymes have an optimum ionic concentration in which it can catalyze a reaction so as the ionic concentration increased the enzyme denatured and the activity decreased. 5. Temperature above 41°C denatured the enzyme. Chemical reactions speed up as the temperature is raised. When temperature increases, more of the reacting molecules have the kinetic energy required to undergo the reaction and tend to go faster with increasing temperature until an optimum temperature is reached. 6. An inhibitor interacts with the enzyme and decreases its activity; therefore, the lesser CuSO4 concentration the greater the enzyme catalase activity. Literature Cited Bartos and Groh. (n.d.). Proceedings of the Society for Experimental Biology and Medicine 37:613-615. Retrieved September 18, 2008, from http://www.enzymeessentials.com/HTM/enzyme _research.html Brookhaven National Laboratory. (2001, December 4). Enzyme Studies at Brookhaven Lab May Lead to New Antiviral Agents. Retrieved September 18, 2008, from http://www.bnl.gov/bnlweb/ pubaf/pr/2001/bnlpr120401.htm Enzymes. (2004, December 9). Retrieved September 18, 2008, from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html Enzyme Essentials. (2005). Selected Enzyme Research. Retrieved September 18, 2008, from http://www.enzymeessentials.com/HTM/enzyme _research.html Howell, E. (1985). Enzyme Nutrition. Avery Publishing Co. Retrieved September 18, 2008, from http://www.enzymeessentials.com/HTM/enzyme _research.html Ophardt, Charles. (2003). Role of Enzymes in Biological Reactions. Retrieved September 18, 2008, from http://elmhcx9.elmhurt.edu/~chm/vchembook/570enzymes.html