Enzyme Catalysis Lab Background: Enzymes catalyze biological systems. They speed up reactions by lowering the activation energy needed for the molecules to begin reacting with each other. Enzymes form enzyme-substrate complexes where the enzyme and the substrate bind at the active site. If the shape of an enzyme is changed in any way, or the protein is denatured then the binding site on the enzyme also changes thus disrupting enzyme function. Enzymes are categorized into two groups, those that are catabolic (break down molecules) and those that are anabolic (build molecules). In this lab you will focus on the enzyme catalase, one which catalyzes hydrogen peroxide into oxygen gas and water. We will use H2SO4 (a strong acid) to stop the reaction in order determine the rate of reaction. After determining the rate of the enzymatic reaction, you will design an experiment to test the effects of a variable on the enzymatic rate of reaction. Pre-Lab Questions: 1. What is the chemical reaction in this lab? Write it in the space below and identify the enzyme, substrate and products in the reaction. 2. If an enzyme and a substrate have a long time to react versus the same enzyme and substrate with less time to react, what would expect your results to be? Question: How does time affect the reaction rates of catalase? Part I: Determine the Baseline Assay 1. 2. 3. 4. In the reaction cup, add 1 ml H2O (pipette), 10 ml of H2O2 (syringe), and 10 ml of H2SO4 (syringe). Swirl. ( NO catalase!) Using the mixture syringe, place 5 ml of the mixture into the titration cup. Titrate the mixture to a persistent brown/pink color. Record the amount (to the nearest hundredth) of KMnO4 needed to titrate the solution. This number indirectly represents the amount of H2O2 in the solution. Amount of KMnO4: ________ Part II: Determine the Rate of Reaction. See data table on the back of this handout 1. 2. 3. 4. 5. 6. 7. In the reaction cup, add 1 ml catalase (pipette) and 10 ml of H2O2. Swirl. Wait 360s and push 10ml of H2SO4 (syringe) into the reaction cup. Swirl. Using the mixture syringe, place 5 ml of the mixture into the titration cup. Titrate the mixture to a persistent brown/pink color. Record the amount in column 3 (to the nearest hundredth) of KMnO4 needed to titrate the solution. Repeat steps 1-5 five more times, changing the wait time (Step 2) to 180s, 90s, 60s, 30s, and 10s. Determine the amount of H2O2 decomposed in each trial by subtracting the amount recorded in in column 4 from the amount recorded in column 2 of the Baseline Assay procedure above. Column 1 Column 2 Time (seconds or s) Baseline (milliliters or ml). The value will be the same in each cell of this column determined from Part 1 Column 3 Amount of KMnO4 needed to titrate solution (milliliters or ml) Column 4 Amount of H2O2 decomposed (Column 2 - Column 3) (no units for this value) 0 10 30 60 90 180 360 8. Determine the rate of reaction for each time interval. Use the following formula: (Amount of H2O2 Decomposed2 - Amount of H2O2 Decomposed1) / (Time2 - Time1) Time (in seconds or s) Rate of Reaction (units of H2O2 decomposed/time) 0 to 10 s 10 to 30 s 30 to 60 s 60 to 90 s 90 to 180 s 180 to 360 s Part III: Design and carry out your group experiment. Think about the following: What are three or four factors that vary in the environment in which organisms live? Which of those factors do you think could affect enzyme activity? How would you modify your basic assay to test your group’s hypothesis?