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Enzyme Catalysis

Introduction Enzymes are proteins produced by living cells that act as catalysts, which affect the rate of a biochemical reaction. They allow these complex biochemical reactions to occur at a relatively low temperature and with less energy usage.

In enzyme--catalyzed reactions, a substrate, the substance to be acted upon, binds to the active site on an enzyme to form the desired product. Each active site on the enzyme is unique to the substrate it will bind with causing each to have an individual three--dimensional structure. This reaction is reversible and is shown as following:

E + S--------ES-------- E + P

Enzymes are recyclable and unchanged during the reaction. The active site is the only part of the enzyme that reacts with the substrate. However, its unique protein structure under certain circumstances can easily be denatured. Some of the factors that affect enzyme reactions are salt concentration, pH, temperature, substrate and product concentration, and activators and inhibitors.

Enzymes require a very specific environment to be affective. Salt concentration must be in an intermediate concentration. If the salt concentration is too low, the enzyme side chains will attract each other and form an inactive precipitate. Likewise, if the salt concentration is too high, the enzyme reaction is blocked by the salt ions. The optimum pH for an enzyme--catalyzed reaction is neutral (7 on the pH scale). If the pH rises and becomes basic, the enzyme begins losing its H+ ions, and if it becomes too acidic, the enzyme gains H+ ions. Both of these conditions denature the enzyme and cause its active site to change shape.

Enzymes also have a temperature optimum, which is obtained when the enzyme is working at its fastest, and if raised any further, the enzyme would denature. For substrate and product concentrations, enzymes follow the law of mass action, which says that the direction of a reaction is directly dependent on the concentration. Activators make active sites better fit a substrate causing the reaction rate to increase. Inhibitors bind with the enzymes' active site and block the substrate from bonding causing the reaction to subside.

The enzyme in this lab is catalase, which produced by living organisms to prevent the accumulation of toxic hydrogen peroxide. Hydrogen peroxide decomposes to form water and oxygen as in the following equation:

2H2O2 ? 2H2O + O2

This reaction occurs spontaneously without catalase, but the enzyme speeds the reaction considerably. This lab's purpose is to prove that catalase does speed the decomposition of hydrogen peroxide and to determine the rate of this reaction.

Hypothesis The enzyme catalase, under optimum conditions, effectively speeds the decomposition of hydrogen peroxide.

Materials Exercise 2A: Test of Catalase Activity

In Part 1, the materials used were 10mL of 1.5% H2O2, 50--mL glass beaker, 1 mL catalase, and 2 10-- mL pipettes and pipette pumps. In Part 2, the materials used were 5 mL of catalase, a boiling water bath, 1 test tube, a test tube rack, 10 mL of 1.5% H2O2, 50--mL beaker, and 2 10--mL pipettes and pipette pumps. In Part 3, the materials used were 10 mL of 1.5% H2O2, 50--mL beaker, liver, and a syringe.

Exercise 2B: The Baseline Assay

This part of the lab required 10 mL of 1.5% H2O2, 1 mL distilled H2O, 10 mL of H2SO4, 2 50--mL beakers, a sheet of white paper, 5 mL KMnO4, 2 5--mL syringes, and 2 10--mL pipettes and pumps.

Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition

The materials used for this section were 15 mL of 1.5% H2O2, 1 mL distilled H2O, 10 mL H2SO4, 2 50-- mL beakers, a sheet of white paper, 5 mL KMnO4, 2 5--mL syringes, and 2 10--mL pipettes and pumps.

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

The materials required for Exercise 2D were 70 mL of 1.5% H2O2, 70 mL of H2SO4, 6 mL of catalase solution, 13 plastic, labeled cups, 3 100--mL beakers, 1 50--mL beaker, 1 10--mL syringe, 1 5--mL syringe, 1 60--mL syringe, a sheet of white paper, a timer, and 30 mL of KMnO4.

Method Exercise 2A: Test of Catalase Activity

In Part 1, 10 mL of 1.5% H2O2 were transferred into a 50--mL beaker. Then, 1 mL of fresh catalase solution was added and the reaction was observed and recorded. In Part 2, 5 mL of catalase was placed in a test tube and put in a boiling water bath for five minutes. 10 mL of 1.5% H2O2 were transferred to a 50--mL beaker and 1 mL of the boiled catalase was added. The reaction was observed and recorded. In Part 3, 10mL of 1.5% H2O2 were transferred to a 50 mL beaker. 1 cm3 of liver was added to the beaker and the reaction was observed and recorded.

Exercise 2B: The Baseline Assay

10 mL of 1.5% H2O2 were transferred to a 50--mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A 5--mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded. The baseline assay was calculated.

Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition

A small quantity of H2O2 was placed in a beaker and stored uncovered for approximately 24 hours. To determine the amount of H2O2 remaining, 10 mL of 1.5% H2O2 were transferred to a 50--mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A 5--mL syringe was used to add

KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded. The percent of the spontaneously decomposed H2O2 was calculated.

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

The baseline assay was reestablished following the directions of Exercise 2B. Before starting the actual experiment a lot of preparation was required. Six labeled cups were set out according to their times and 10 mL of H2O2 were added to each cup. 6 mL of catalase were placed in a 10--mL syringe, and 60 mL of H2SO4 were placed in a 60--mL syringe. To start the actual lab, 1 mL of catalase was added to each of the cups, while simultaneously, the timer was started. Each of the cups were swirled. At 10 seconds, 10 mL of H2SO4 were added to stop the reaction. The same steps were repeated for the 30, 60, 120, 180, and 360 second cups, respectively.

Afterwards, a five 5 mL sample of each of the larger cups were moved to the corresponding labeled smaller cups. Each sample was assayed separately by placing each over a white sheet of paper. A 5-- mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded.

Results

Activity

Observations

Enzyme activity

The solution only bubbled slightly and slowly.

Effect of Extreme temperature

The catalase had no reaction with the H2O2; there were no bubbles

Presence of catalase

The solution foamed up immediately

Table 1 Enzyme Activity

Table 2 Establishing a Baseline

Volume

Initial reading

5.0 mL

Final reading

0.8 mL

Baseline ( final volume ? initial volume)

4.2 mL

Table 3 Rate of Hydrogen Peroxide Spontaneous Decomposition

Volume

Initial KMnO4

5.0 mL

Final KMnO4

1.2 mL

Amount of KMnO4 used after 24 hours

3.8 mL

Amount of H2O2 spontaneously decomposed ( ml baseline ? ml after 24 hours)

0.4 mL

Percent of H2O2 spontaneously decomposed ( ml baseline ? ml after 24 hours/ baseline)

9.52%

Table 4 Rate of Hydrogen Peroxide Decomposition by Catalase

Time ( Seconds)

Baseline KMnO4

10 4.0 mL

30 4.0 mL

60 4.0 mL

120 4.0 mL

180 4.0 mL

360 4.0 mL

Initial volume 5.0 mL KMnO4

5.0 mL

5.0 mL 5.0 mL

5.0 mL

5.0 mL

Final volume KMnO4

2.2 mL

1.4 mL

2.0 mL

1.7 mL

2.4 mL

2.3 mL

Amount KMnO4 used (baseline ? final)

2.8 mL

3.6 mL

3.0 mL

3.3 mL

2.6 mL

2.7 mL

Amount H2O2 used (KMnO4 ? initial)

1.2 mL

0.4 mL

1.0 mL

0.7 mL

1.4 mL

1.3 mL

Amount of Hydrogen Peroxide Decomposed by Catalase

Exercise 2A: Test of Catalase Activity

1. Observing the reaction of catalase on hydrogen peroxide:

a. What is the enzyme in this reaction?

catalase

b. What is the substrate in this reaction? Hydrogen peroxide

c. What is the product in this reaction? Oxygen & water

d. How could you show that the gas evolved is O2? The gas could be shown to be O2 if the gas were collected in a tube, and a glowing splint was held in the tube. If the splint glowed, it would prove the gas was oxygen.

2. Demonstrating the effect of boiling on enzyme action:

a. How does the reaction compare to the one using the unboiled catalase? Explain the reason for this difference. While the unboiled catalase caused bubbles to form in the solution, the boiled catalase did not react at all because boiling an enzyme causes the protein to unfold and therefore denatures it.

3. Demonstrating the presence of catalase in living tissue:

a. What do you think would happen if the potato or liver was boiled before being added to the H2O2? The catalase in the liver would have been denatured by the boiling and would not have reacted with the H2O2.

Analysis of Results

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