AP Laboratory #2: Enzyme Catalysis

Problem
How will enzyme catalase react under varying conditions using temperature and pH over a course of time?

Background
Enzymes are proteins that are produced by living things as well as biochemical catalysts that are capable of lowering the activation energy needed for a biochemical

reaction to occur. As a result of enzyme activity, cells are able to carry out complex chemical activities at relatively low temperatures. The substrate is the substance acted upon in an enzyme-catalyzed reaction, and it can bind reversibly to the active site of the enzyme. An active site is the part of the enzyme that interacts with the substrate so that any substrate that blocks or changes the shape of the active sit affects the activity of the enzyme. This however is only temporary union and it reduces the amount of energy that is needed to activate the reaction of the substrate molecule causing products to be formed. Equation shows this process: E + S ES E + P Enzymes follow the law of mass reaction. This means that the enzyme is not changed in the reaction and can be recycled to break down additional substrate molecules. There are multiple factors that can affect the action of an enzyme such as: temperature, the pH of the environment, salt concentration, activations and then inhibitors. Chemical reactions usually speed up as the temperature increases. The reacting molecules have enough kinetic energy to go through the reaction while the temperature increases. But if the temperature goes above the optimal temperature, then the conformation of the enzyme molecules is disrupted. Then for pH, it has a scale that measures the acidity or H+ concentration in a solution and goes from 0 to 14, with 0 being the most acidic and 14 least. Amino acid side chains have groups such as COOH groups that can gain or lose H+ ions. When the pH is lowered then an enzyme will usually gain H+ ions, causing a disruption in the enzymes shape. While on the other hand, if the pH is raised, then enzyme will lose H+ ions and lose its active shape. Reactions can perform usually at its best in neutral environments. Next would be salt concentration. If it is close to zero, then the changed amino acid side chains of the enzyme molecules will become attracted to one another. The enzyme denatures and will then form an inactive precipitate. This usually happens when a lot of heat destroys the structure of proteins. This usually occurs at 40 to 50 Celsius. If the concentration is high, then the normal interaction of charged groups will become blocked. Normally the optimum for enzyme activity is an intermediate salt concentration. An example of intermediate concentrations would be the salt concentration of blood and cytoplasm. An activator is a coenzyme that can increase the rate of a reaction and it can also regulate the speed at which an enzyme acts. Also, it makes the active site a better fit for the substrate. An inhibitor has somewhat a similar power as the activator regulation except that it decreases the reaction rate. In this lab we use the enzyme catalase, which has four polypeptide chains that are each made up of more than 500 amino acids. One of its functions would be that it can prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of

metabolic processes. There are many oxidation reactions that occur in cells that involve catalase. The decomposition of hydrogen peroxide to form water and oxygen is the primary reaction catalyzed by catalase: 2 H2O2 2 H2O + O2 (gas) Without catalase this reaction occurs spontaneously but very slowly. Catalase speeds up the reaction. The direction of an enzyme-catalyzed reaction is dependent on the concentration of enzyme, substrate, and product. Through this lab we learn how to measure the amount of product formed, the amount of substrate used, from the moment the reactants are brought together until the reaction has been stopped.

Hypothesis
While working under optimum conditions with the enzyme catalase, there are noticeable increases in the rate that hydrogen peroxide will decompose.

Procedure
General Procedure 1. A purified catalase extract is mixed with substrate (H2O2) in a beaker. The enzyme catalyzes the conversion of H2O2 to H2O and O2 (gas). 2. Before all the H2O2 is converted to H2O2 and O2, the reaction is stopped by adding sulfuric acid (H2SO4). The H2SO4 lowers the pH, denatures the enzyme and thereby stops the enzymes catalytic activity. 3. After the reaction is stopped, the amount of substrate (H2O2) remaining in the beaker is measures. To assay (measure) this quantity, potassium permanganate is used. Potassium permanganate (KMnO4), in the presence of H2O2 and H2SO4 reacts as follows: 5 H2O2 + 2 KMnO4 + 3 H2SO4 K2SO4 + 2 MnSO4 + 8 H2O + 5 O2 Establishing Baseline 1. Put 10 mL of 1.5% H2O2 into a clean glass beaker. 2. Add 1 mL of H2O (instead of enzyme solution). 3. Add 10 mL of H2SO4 (1.0 M). USE EXTREME CARE IN HANDLING ACIDS. Your teacher will instruct you about the proper safety procedures for handling hazardous materials. 4. Mix well.

5. Remove a 5 mL sample. Place this 5 mL in another, and assay for the amount of H2O2 as follows. Place the beaker containing the sample over a white paper. Use burette of 5 mL pipette to add KMnO4 a drop at a time to the solution until a persistent pink or brown color is obtained. Remember to gently swirl the solution after adding each drop. Check to be sure that you understand the calibrations on the burette or syringe.

Exercise 2D
1. To determine the course of an enzymatic reaction, how much substrate is disappearing over time must be measured. First, set up the cups with the times and the word acid up. 2. Add 10 mL of H2SO4 to each of the cups marked acid. 3. Then put 10 mL of 1.5% H2O2 into the cup marked 10 sec. 4. Add 1 mL of catalase extract to this cup. 5. Swirl gently for 10 seconds. (Calculate time using the timer for accuracy.) 6. At 10 seconds, add the contents of one of the acid filled cups. 7. Remove 5 mL and place in the second cup marked 10 sec. 8. Assay the 5-mL sample by adding KMnO4 a drop at a time until the solution obtains a pink or brown color. 9. Repeat the steps 2 through 8 except allow the reactions to be timed for 30, 60, 120, 180, and 360 seconds. Using the times corresponding, marked cups. Record all results and observations.

Data and Results Exercise 2B: The Baseline Assay

Baseline Calculation Final reading of burette Initial reading of burette Baseline (Final- Initial) 4.3 mL 0.3 mL 4 mL KMnO4

Exercise 2C: The Uncatalyzed Rate of H2O2

Uncatalyzed H2O2 decomposition Final reading of burette 2 mL

Initial reading of burette Amount of KMnO4 titrant Amount of H2O2 spontaneously decomposed What percent of the H2O2 spontaneously decomposes in 24 hours?

1.6 mL 4 mL 0 mL 60 %

Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition

Baseline Calculation Final reading of burette Initial reading of burette Baseline (Final Initial) 11.8 mL 4.3 mL 7.5 mL KMnO4

KMnO4 A. Baseline B. Final Reading C. Initial Reading D. Amount of KMnO4 consumed E. Amount of H2O2 used

Time-Course Determination Time (Seconds) 10 30 60 120 4 4 4 4 11.8 18.8 19.5 20.0 4.3 11.8 18.8 19.5 7.5 7 .7 .5 3.5 3 3.3 3.5

180 4 20.1 20.0 .1 3.9

360 4 20.3 20.1 .2 3.8

Graph the data for enzyme-catalyzed H2O2 decomposition a. the independent variable____________Time in Seconds___________________ Use this to label the horizontal (X) axis. b. the dependent variable__________Amount of Hydrogen Peroxide (mL)_______ Use this to label the vertical (Y) axis.

Amount of Hydrogen Peroxide Decomposed by Catalase

4.5 4

Amount of Hydrogen Peroxide (mL)

3.5 3 2.5 2 1.5 1 0.5 0 10 30 60 120 180 360

Time in seconds

Conclusion Exercise 2A: Test of Catalase Activity

1. A) What is the enzyme in this reaction? The enzyme in this reaction is catalase solution. B) What is the substrate in this reaction? The substrate is hydrogen peroxide. C) What is the product in this reaction? The products are water and oxygen gas. D) How could you show that the gas evolved is O2? You could show that the gas evolved is O2 by referring to the equation 2H2O2 + Catalase solutionH2O + O2, where the only gas released is oxygen. 2. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference. Compared to the one using the unboiled catalase, the one with the boiled catalase didnt have any signs of bubbling because the catalase had been denatured by the heat and resulting in no reaction.

3. What do you observe? What do you think would happen if the liver were boiled before being added to the hydrogen peroxide? I observe that there is a lot of gas being released from the solution. If the liver were boiled before being added to the hydrogen peroxide I think that there wouldnt be any signs of a reaction since the catalase that occurs naturally within the liver would have already been denatured preventing the reaction from occurring.

Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition

1.) Reaction rate (mL H2O2 / sec)

Time Intervals (Seconds)

Rates Initial 0 to 10 .35 mL/sec 10 to 30 -.025 mL/sec 30 to 60 .01 mL/sec 60 to 120 .0033 mL/sec 120 to 180 .00667 mL/sec 180 to 360 -5.556 E -4 mL/sec

2.) When is the rate the highest? Explain why. The rate is the highest in the first ten seconds of the experiment because the rate is decreasing as the concentration of the catalase is decreasing over time. 3.) When is the rate the lowest? For what reasons is the rate low? The rate is the lowest during the 180 to 360 seconds time period because of the law of mass action. This law states that when there is a great/high concentration of a product like there was in this time interval, the enzymes will be blocked by the product, which in this case would be water from reacting or even getting to the substrate, being H2O2. 4.) Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this to enzyme structure and chemistry The inhibiting effect of sulfuric acid on the function of catalase is that it causes the pH level in the solution to lower a great amount. Acidic solutions cause the protein structure of the enzyme to gain H+ ions, as a result it causes it to denature. 5.) Predict the effect lowering the temperature would have on the rate of enzyme activity. Explain your prediction.

I predict that by lowering the temperature of the catalase would result in slowing down the rate of reaction that is until it causes the enzyme to denature. When this happens it means that it is no longer capable of reacting with the substrate. Lowering the temperature of the catalase would slow the rate of reaction until it finally caused the enzyme to denature, and it would no longer react with the substrate. It would seem that most enzymes are only affective in a temperature that is in a range between 40 - 50 C. 6.) Design a controlled experiment to test the effect of varying pH, temperature, or enzyme concentration. Part 1: Enzyme Activity at Room Temperature Put 10 mL of 1.5% H2O2 in a 50-mL beaker, and then add 1 mL of room temperature catalase. Mix well and add 10 mL of H2SO4. Look for the reaction and record the data. Part 2: The Effect of Excessive Heat on Enzyme Activity Add 5 mL of catalase to a test tube and then heat it over a Bunsen burner. Add 1 mL of the heated catalase to 10 mL of 1.5% H2O2 in a 50-mL beaker. Then you would put 10 mL of H2SO4. Look for the reaction and record the data. Part 3: The Effect of Excessive Cooling on Enzyme Activity Put 5 mL of catalase in a freezer until it gets completely frozen. Add 1 mL of the frozen catalase to 10 mL of 1.5% H2O2 in a 50-mL beaker. Add 10 mL of H2SO4. Look for the reaction and record the data