David Solinger AP Biology November 22, 2011 Lab 2: Enzyme Catalysis Objectives: Measure the effects of changes in temperature,

e, PH, enzyme concentration, and substrate on reaction rates of an enzyme-catalyzed reaction in a controlled experiment Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Introduction: Enzymes catalyze reactions by lowering the activation energy necessary for a reaction to occur. The molecule that an enzyme acts on is called the substrate. In an enzyme-mediated reaction the enzyme molecule is unchanged after the reaction, and it can continue to catalyze the same type of reaction over and over. Each enzyme is specific for the reaction it will catalyze. Enzymes are globular proteins. Their structure creates an area known as the active site. Active sites are specific to only one type of substrate. Only substrates that have the specific shape are able to bind with the enzyme's active site. When an enzyme binds to a substrate, the active site changes slightly. This change is known as an induced fit. Induced fit creates a tighter fit between the substrate and enzyme, strengthening the bonds between them. Enzymes are sensitive to changes in the enzyme's environment. Important changes include salt concentration, pH, and temperature. Enough variation in salt concentration, pH, or temperature can cause the enzyme to no longer catalyze reactions. An enzyme is said to be denatured when it loses its functional shape. Each enzyme functions best within a certain pH and temperature range. When the pH or temperature changes, the shape of the enzyme and active site can become distorted and affects enzyme function. Generally chemical reactions speed up as temperature is increased, but each enzyme has a temperature where the enzyme's functional shape is lost. Inhibitors can prevent an enzyme from catalyzing a specific reaction. There are two types of inhibitors: allosteric inhibitors and competitive inhibitors. An allosteric inhibitor binds to an enzyme at an allosteric site, (any site other than the active site) and as a result inhibits the enzymes activity. A competitive inhibitor competes directly with a substrate at an active site to impede enzyme activity. The enzyme used in this lab is catalase. Catalase catalyzes the denaturation of hydrogen peroxide into oxygen gas and water. In absence of catalase the reaction continues but catalase speeds up the reaction significantly. Catalase is present in organisms in order to prevent a toxic buildup of hydrogen peroxide. We are going to measure the rate at which the reaction proceeds when catalase is involved. We will use H2SO4 to stop the reaction from proceeding, because it denatures the enzyme catalase as a result of the change in pH.

Part A: Test of Catalase Activity 1) a) The enzyme is catalase. b) The substrate is hydrogen peroxide. c) The products are oxygen gas and water. d) To determine if the gas is oxygen gas you could first capture the gas in an upside down beaker or something similar. Then do the splint test by lighting a splint, blowing it out, and placing it in the upside down beaker. If the splint glows red or relights that means a high concentration of oxygen is present. 2) The boiled catalase did nothing to the hydrogen peroxide because the enzyme was denatured as a result of the high temperature when it was boiled. 3) When the liver was added to the hydrogen peroxide bubbles were observed, most likely oxygen gas resulting from the denaturation of the hydrogen peroxide. I think if the liver was boiled the catalase present in the liver would be denatured, so nothing would happen if it were added to the hydrogen peroxide. Part B: The Base Line Assay Base line calculation Final reading of the burette Initial reading of the burette Base line (Final Initial)

41.7 mL 38.1 mL 3.6 mL of KMnO4

Part C: The Uncatalyzed Rate of H2O2 Decomposition Uncatalyzed H2O2 Decomposition Final reading of the burette 41.7 mL Initial reading of the burette 38.1 mL Amount of KMnO4 titrant 3.6 mL of KMnO4 Amount of H2O2 spontaneously decomposed (mL baseline mL KMnO4 0.4 mL What percentage of the H2O2 spontaneously decomposes in 24 hours? [(mL baseline mL KMnO4)/ mL baseline] x 100 11.1 %

Part D: An Enzyme-Catalyzed Rate of H2O2 Decomposition Base line calculation Final reading of the burette Initial reading of the burette Base line (Final Initial)

47.4 mL 44.6 mL 2.8 mL of KMnO4

Table 2.1: Timed Enzyme-Catalyzed H2O2 Decomposition KMnO (mL) a) Base Line b) Final Reading c) Initial Reading d) Amount of KMnO Consumed (B minus C) e) Amount of HO Used (A minus D) Time (seconds) 30 60 90 120 180 2.8 2.8 2.8 2.8 2.8 39.2 40.7 42.8 43.9 44.6 37.3 39.2 41.5 42.8 43.9 1.9 1.5 1.3 1.1 0.7 0.9 1.3 1.5 1.7 2.1

10 2.8 37.3 34.8 2.5 0.3

Graph 2.1: Amount of HO Used Versus Time The independent variable is time and the dependent variable is the amount of HO decomposed.

Amount of HO Used Versus Time

2.5

Amount of HO Used (mL)

1.5

0.5

0 0 20 40 60 80 100 120 140 160 180 200

Time (seconds)

Analysis of Results 1) Rate of HO Used Time Intervals (seconds) Initial 0 to 10 Rates (mL HO/sec) 0.03 10 to 30 0.03 30 to 60 0.013 60 to 90 0.0067 90 to 120 0.0067 120 to 180 0.0067

2) The rate of HO used is the greatest at the beginning, because that is when there is the most substrate available to react and the least amount of products to impede the enzyme and substrate from interacting. 3) The rate of HO used is the least the longer the reaction is allowed to take place, because as time passes more of the HO is being used up in the reaction leaving less to react with the enzyme. In addition to the less HO available it is also less concentrated because water is being produced as a product of the reaction. 4) The sulfuric acid denatures the catalase, preventing it from reacting with the hydrogen peroxide. Sulfuric acid lowers the pH of catalases environment beyond the pH range in which catalase can operate. The acidic environment causes the structure of catalase to be destroyed rendering it ineffective in catalyzing hydrogen peroxides denaturation. 5) I predict lowering the temperature would slow down the enzyme activity. The bonds within catalase will still stay intact, but a lower temperature of the solution means there will be a lower average kinetic energy of the molecules within the solution. The reaction will slow down as a result of the decreased amount of molecular collisions. If the temperature is decreased enough the reaction may stop completely if no molecules can reach the activation energy. 6) To test the effect of temperature on enzyme activity start with three beakers filled with 10 mL of 1.5% HO solution. Prepare an ice water bath, a room temperature water bath, and a hot water bath. Place one of the beakers in each of the baths. After the beakers have sat for five minute in their respective baths add 1 mL of catalase. 60 seconds after the catalase has been added, add 10mL of sulfuric acid to the three beakers to denature the catalase. Then, using a burette, titrate KMnO into the beakers to determine how much hydrogen peroxide was decomposed. Ideally as temperature increases the amount of hydrogen peroxide decomposed in 60 seconds should increase.