Week 4 Ch 4 Lab 7 Enzymes

Note: All your answers to questions must be in Red or other color (not including blue) for easier grading. Points will be deducted if you do not distinguish your answers. Lab 7. Enzymes Objectives:  Define the following terms: metabolism, reactant, product, substrate, enzyme, denature  Describe the specific action of the enzyme catalase, include the substrate and products of the reaction  List the factors that can affect the rate of a chemical reaction and enzyme activity  Explain why enzymes have an optimal pH and temperature to ensure greatest activity (greatest functioning) of the enzyme (be sure to consider how virtually all enzymes are proteins and the impact that temperature and pH may have on protein function)  Explain why the same type of chemical reaction performed at different temperatures revealed different results/enzyme activity  Explain why warm temperatures (but not boiling) typically promote enzyme activity but cold temperature typically decreases enzyme activity  Explain why increasing enzyme concentration promotes enzyme activity  Explain why the optimal pH of a particular enzyme promotes its activity Vocabulary:  Metabolism  Reactant  Product  Substrate  Enzyme  Catalyst  Denature  Activation Energy Introduction: Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy necessary to break the chemical bonds in reactants and form new chemical

bonds in the products . Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life. The functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of the reactant (s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called substrates . The active site of an enzyme recognizes, confines, and orients the substrate in a particular direction. Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example, proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by the enzyme amylase). Notice that both enzymes end in the suffix -ase. This suffix indicates that a molecule is an enzyme. Environmental factors may affect the ability of enzymes to function. You will perform a set of experiments to examine the effects of temperature, pH, and substrate concentration on the ability of enzymes to catalyze chemical reactions. You will be examining the effects of these environmental factors on the ability of catalase to convert H 2 O 2 into H 2 O and O 2 . Part 1: Testing Catalase Activity Background Information: Hydrogen peroxide is a toxic product of many chemical reactions that occur in living things. Although it is produced in small amounts, living things must detoxify this compound and break down hydrogen peroxide into water and oxygen, two non-harmful molecules. The organelle responsible for destroying hydrogen peroxide is the peroxisome using the enzyme catalase. Both plants and animals have peroxisomes with catalase. The catalase sample for today’s lab will be from a potato. Question: How do we know catalase is present in the test tubes? Hypothesis: Test tube A will have a rapid formation of bubbles. Materials:  Two clean test tubes  Test tube rack  Ruler  1 mL graduated pipet  Catalase (from potato)  Hydrogen Peroxide  Distilled water (dH 2 O)

Each enzyme has an optimum temperature at which it works best. A higher temperature generally results in an increase in enzyme activity . As the temperature increases, molecular motion increases resulting in more molecular collisions. If, however, the temperature rises above a certain point, the heat will denature the enzyme, causing it to lose its three-dimensional functional shape by denaturing its hydrogen bonds. Cold temperature, on the other hand, slows down enzyme activity by decreasing molecular motion. In this experiment, you will explore this effect on the catalase extracted from a potato. Question: How does temperature affect the rate of enzyme activity? Hypothesis: At 0°C, catalase activity occurs at a faster rate Materials:  Three clean test tubes  Two 5 mL Graduated pipet  Hydrogen peroxide  Catalase (potato)  Ruler  Ice bath  37°C and 100°C water bath  Thermometer  Boiling water  Wax pencil Procedure:  Label each of your test tubes (T1, T2, and T3).  To each of your test tubes add 1 mL of catalase.  Place T1 in an ice bath and start your first timer for 15 minutes. Record your starting temperature in Table 2 .  Wait 5 minutes and then place T2 in a 37°C water bath and start your second timer for 15 minutes. Record your starting temperature in Table 2 .  Wait an additional 5 minutes after placing T2 in the 37°C water bath before placing T3 in the 100°C water bath and start your third timer for 15 minutes. Record your starting temperature in Table 2 .  As soon as the timer rings, take out T1 out of the ice bath and add 4 mL of hydrogen peroxide.  Carefully, swirl the mix and wait 30 seconds.  Measure the bubbles height (mm) and record your results in Table 2 .  Repeat steps for the remaining two test tubes when their timer rings. Results: (Watch the Lab 7. Enzymes video)

Table 2: Effects of Temperature in Enzyme Activity Test Tube Temperature (°C) Bubble Height (mm) 1 0°C 2.2 mm 2 37°C 1.3 mm 3 100°C 0.2 mm Create a bar graph using the data in excel for your data and paste it below. (Be sure to label your axes.) Conclusion: 5. Explain your results for Test tube 1. From sitting in an ice bath at 0°C to adding the hydrogen peroxide, and measuring the bubble reaction, it continued to expand rapidly. I could see the bubbles in full-speed conversation (bubbling) at a fast rate. It reminds me of a carbonated beverage. A higher temperature resulted in an increase in enzyme activity.

 Ruler  Wax pencil Procedure:  In order to test enzyme concentration, we will be diluting the catalase with water. Use the beakers to dilute the catalase. Mix according to the table below. Beaker Water Catalase Concentration 1 3 mL of water 1 mL of Catalase 25% 2 2 mL of water 2 mL of Catalase 50% 3 1 mL of water 3 mL of Catalase 75% 4 0 mL of water 4 mL of Catalase 100%  Label each of your test tubes (E1, E2, E3, and E4).  To each of your test tubes add 4 mL of hydrogen peroxide.  To E1, add 1 mL of 25% Catalase. Carefully swirl the mix, wait 30 seconds, and record the bubble height (mm) in Table 3.  To E2, add 1 mL of 50% Catalase. Carefully swirl the mix, wait 30 seconds, and record the bubble height (mm) in Table 3.  To E3, add 1 mL of 75% Catalase. Carefully swirl the mix, wait 30 seconds, and record the bubble height (mm) in Table 3.  To E4, add 1 mL of 100% Catalase. Carefully swirl the mix, wait 30 seconds, and record the bubble height (mm) in Table 3. Results: (Watch the Lab 7. Enzymes video) Table 3: Effects of Enzyme Concentration Test Tube Catalase Concentration Bubble Height (mm) Class Average Bubble Height (mm) E1 1 ml of 25% 0.2 mm N/A E2 1 ml of 50% 0.6 mm N/A E3 1 ml of 75% 3.2 mm N/A E4 1 ml of 100% 3.0 mm N/A Create a bar graph using the data in excel for your data and paste it below. (Be sure to label your axes.)

Conclusion: 9. Explain your results for Test tube E1. At 3mL of water to 1mL of the Catalase bringing the concentration to 25%, the bubble reaction was inactive and low. Looking at it at the end of the video, the bubbles are a little larger than before but are still at the low end of the E2, E3, and E4 test tubes, yet I can see them bubbling at the bottom. 10. Explain your results for Test tube E2. At 2mL of water to 2mL of the Catalase bringing the concentration to 50%, the bubble reaction is slowly rising as the bubbles are congregating. I can see the bubbles moving fast. Looking at it at the end of the video, the bubble reaction stretched more than the E1 and E4 test tubes. 11. Explain your results for Test tube E3. At 1mL of water to 3mL of the Catalase bringing the concentration to 75%, the bubble reaction is fast at work, bubbling and climbing at a rapid rate than E1 and E2 test tubes. By the end of the video, those bubbles are still going.

 Add 4 mL of hydrogen peroxide to all three test tubes.  Carefully swirl the mix, wait 30 seconds, and record the bubble height (mm) in Table 4. Results: (Watch the Lab 7. Enzymes video) Table 4: Effects of pH in Enzyme Activity Test Tube pH Bubble Height (mm) Class Average Bubble Height (mm) P1 3 0.5 mm N/A P2 7 1.5 mm N/A P3 11 1.0 mm N/A Create a bar graph using the data in excel for your data and paste it below. (Be sure to label your axes.) Conclusion: 14. Explain your results for Test tube P1. Observed decrease in bubble height at pH 3 for Test Tube P1 which is likely due to the denaturation of the enzyme since it is acidic. 15. Explain your results for Test tube P2. The bubble height in Test Tube P2 was a lot higher than that of Test Tube P1 because the enzyme is affected by changes in pH, so from pH

3 to pH 7 it was quite noticeable. 16. Explain your results for Test tube P3. Shrinking of bubble height in Test Tube P3 with pH 11 after reaching a height point, top bubbles began to pop. Once again denaturation played a role here since the pH value is high therefore the enzyme activity is at a loss. 17. What is the optimum pH for your enzyme to function? The ideal pH range is at 7. Enzymes have an optimal pH range at which they function most effectively, but deviating from the optimal pH can disrupt the enzyme’s structure by reducing its activity; therefore, changing shape by denaturation. 18. In mostly all the experiments, you were asked to find the class average bubble height, why do you think this was for? As enzyme concentration increases so does bubble height signifying enzyme activity is higher and bubble height changes due to how time affects enzyme activity. Licenses and Attributions:  " Enzymes " by Lumen Learning , LibreTexts is licensed under CC BY 4.0 .  " Enzymes " by Gary Kaiser , LibreTexts is licensed under CC BY .  " Investigation: Enzyme and Substrate Concentrations " by LibreTexts is licensed under CC BY-NC-SA 4.0 .