Enzyme Catalysis Dry Lab

An experiment was done to measure the rate of an enzyme catalyzed raction. The enzyme catalase catalyzes the conversion of H₂O₂ to H₂O and O₂ (gas). The disappearance of the substrate is measured as follows:

1. A purified catalase extract is mixed with substrate (H₂O₂) in a beaker. This experiment determined the rate at which a 1.5% H₂O₂ solution decomposes when catalyzed by the purified catalase extract. The amount of H₂O₂ consumed after 10, 30, 60, 120, 180, and 360 seconds was measured.

2. Before all of the H₂O₂ was converted to H₂O and 0₂, the reaction was stopped by adding sulfuric acid (H₂SO₄). The H₂SO₄ lowers the pH, denatures the enzyme, and thereby stops the enzyme's catalytic activity.

3. After the reaction is stopped, the amount of substrate (H₂O₂) remaining in the beaker was measured. To assay (measure) this quantity, potassium permanganate was used. Potassium permanganate (KMnO₄), in the presence of H₂O₂ and H₂SO₄, reacts as follows:

5H₂O₂ + 2KMnO₄ + 3H₂SO₄ ➝ K₂SO₄ + 2MnSO₄ + 8H₂O + 5O₂

Note that H₂0₂ is a reactant for this reaction. Once all the H₂O₂ has reacted, any more KMnO₄ added will be in excess and will not be decomposed. The addition of excess KMnO₄ causes the solution to have a permanent pink or brown color. Therefore, the amount of H₂O₂ remaining is determined by adding KMnO₄ until the whole mixture stays a faint pink or brown, permanently. This technique is called titration. The amount of KMnO₄ added is a proportional measure of the amount of H₂O₂ remaining (2 molecules of KMnO₄ reacts with 5 molecules of H₂O₂ as shown in the equation).

The difference between the initial and final readings shows how much H₂O₂ is left after the enzyme catalyzed reaction. The shorter the time, the more H₂O₂ remains and therefore the more KMnO₄ necessary to titrate to the endpoint.

The Baseline Assay

To determine the amount of H₂O₂ initially present in a 1.5% solution, one needs to perform all the steps of the procedure without adding catalase to the reaction mixture. This amount is known as the baseline and tells us the initial concentration of H₂O₂ in solution.

Table 1: Burette readings (mL)

Questions

1. Basically, how are we measuring the enzyme activity?

2. Why are we adding H₂SO₄? Why does this have the effect that it does?

3. What is the enzyme, substrate, and product in the reaction?

4. Predict the effect of boiling the catalase before the reaction. Explain.

5. Graph the sample data for the amount of H₂O₂ remaining. What is the independent variable? What is the dependent variable?

6. Why are we able to use the amount of KMnO₄ as a measure of the amount of H₂O₂?

7. During which time interval was the rate of enzyme activity the highest? Explain why.

8. Explain the inhibiting effect of sulfuric acid on the function of catalase. Relate this to enzyme structure and chemistry.

9. Predict the effect that lowering the temperature would have on the rate of enzyme activity. Explain your prediction.

10. Design a controlled experiment to test the effect of varying either pH, temperature, or enzyme concentration.