03.05 Enzyme Action: pH
Overview of pH and Enzyme Activity
- Optimal pH: Each enzyme has a specific pH at which it functions best, known as its optimum pH.
- Most enzymes have an optimum around pH 7 (neutral).
- Example: Pepsin (a protease in the stomach) has an optimum pH of 1.5, suited to the stomach’s acidic environment.
- Impact of Deviating from Optimum pH:
- Too acidic or too alkaline environments alter the enzyme’s 3D structure, particularly affecting the active site.
- Changes in pH disrupt ionic bonds between R groups of amino acids in enzymes, which alters active site shape and can reduce or stop enzyme function.
- Extreme pH levels can denature enzymes by permanently altering their structure.
Mechanism of pH Effect on Enzymes
- Hydrogen Ions (H⁺):
- pH measures H⁺ concentration (low pH = high H⁺).
- H⁺ ions interact with charged R groups in amino acids, potentially disrupting ionic bonding in the enzyme.
- Result: Altered active site shape affects substrate binding, reducing reaction rate or stopping it entirely.
Experimenting with pH in Enzyme Reactions
- Buffer Solutions:
- Buffers maintain a stable pH in reactions, even if the reaction itself would change the pH.
- To test enzyme activity at various pH levels, use different buffers set to the desired pH levels.
- Add a measured volume of buffer to each reaction mixture to keep conditions consistent.
Sample Experiment: Effect of pH on Trypsin Activity
Objective: Investigate how pH affects trypsin’s ability to digest protein in a milk suspension.
Materials:
- Milk powder suspension (4 g in 100 cm³ water)
- 0.5% trypsin solution
- Buffer solutions at different pH levels
- Colorimeter or visual observation setup for clarity measurement
Procedure:
Prepare Reactions:
- Mix equal volumes of milk suspension and trypsin solution.
- Add a specific pH buffer to each mixture to set the reaction at different pH values.
Start Reactions:
- Add trypsin and immediately start timing.
Measure Reaction Rate:
- Record the time taken for the milk suspension to turn clear (protein is digested by trypsin).
- Alternatively, use a colorimeter to measure clarity changes over time.
Record Results:
- Plot rate of reaction vs. pH to determine the optimum pH for trypsin.
Questions & Practical Applications
Effect of Temperature on Catalase:
- Set up experiments at different temperatures to observe catalase’s breakdown rate of hydrogen peroxide.
Protease Use in Washing Powders:
- a) Proteases break down protein-based stains (e.g., blood).
- b) Low temperatures prevent enzyme denaturation, maintaining effectiveness.
- c) High-temperature-resistant proteases allow efficient cleaning in hot water.
- Investigating pH Effect on Trypsin:
- Prepare milk suspensions with trypsin at various pH levels using buffers to find the optimum pH for protein digestion in milk.
Practise Questions
Question 1
Explain how pH affects the 3D structure of an enzyme and its activity. (5 marks)
Mark Scheme:
- Each enzyme has an optimum pH at which it functions best. (1 mark)
- Deviations from the optimum pH disrupt ionic bonds in the enzyme’s 3D structure. (1 mark)
- Changes in the ionic environment alter the shape of the active site, reducing its ability to bind the substrate. (1 mark)
- At extreme pH levels, the enzyme becomes denatured, permanently losing its function. (1 mark)
- This is caused by interactions of H⁺ ions with charged R groups, disrupting ionic bonding. (1 mark)
Question 2
Describe the role of buffers in experiments investigating the effect of pH on enzyme activity. (4 marks)
Mark Scheme:
- Buffers maintain a stable pH in the reaction mixture. (1 mark)
- They ensure that pH changes caused by the reaction itself do not affect results. (1 mark)
- Different buffers set the reaction mixture to specific pH values to test enzyme activity at various pH levels. (1 mark)
- This allows the determination of the enzyme’s optimum pH under controlled conditions. (1 mark)
Question 3
Design an experiment to investigate the effect of pH on trypsin activity using a milk suspension. (6 marks)
Mark Scheme:
- Prepare materials: Milk suspension, 0.5% trypsin solution, and buffers of different pH values. (1 mark)
- Mix equal volumes of milk suspension and trypsin solution in separate test tubes for each pH value. (1 mark)
- Add the corresponding buffer to each test tube to set the reaction at specific pH levels. (1 mark)
- Start timing when trypsin is added, and observe the milk suspension for clarity changes. (1 mark)
- Measure the time taken for the milk to turn clear or use a colorimeter to record clarity changes quantitatively. (1 mark)
- Plot a graph of reaction rate against pH to determine the optimum pH for trypsin. (1 mark)
Question 4
Why do different enzymes have different optimum pH values? Use pepsin and trypsin as examples. (5 marks)
Mark Scheme:
- The optimum pH reflects the natural environment where the enzyme functions. (1 mark)
- Pepsin, found in the stomach, has an optimum pH of 1.5, suited to the acidic environment of gastric juice. (1 mark)
- Trypsin, active in the small intestine, has an optimum pH around 8, suited to the slightly alkaline conditions of the intestine. (1 mark)
- Differences in pH environments ensure enzymes are specialized for their specific roles. (1 mark)
- These adaptations prevent enzymes from being active in inappropriate regions, avoiding unnecessary reactions. (1 mark)
Question 5
Explain why extreme pH levels lead to enzyme denaturation. (4 marks)
Mark Scheme:
- Extreme pH levels cause high concentrations of H⁺ or OH⁻ ions. (1 mark)
- These ions interact with charged R groups in amino acids, disrupting ionic bonds. (1 mark)
- This alters the enzyme’s 3D structure, including the shape of the active site. (1 mark)
- Denatured enzymes lose their ability to bind substrates, and reaction rates drop to zero. (1 mark)
Question 6
Describe how you could measure the clarity of a milk suspension to determine the rate of trypsin activity. (5 marks)
Mark Scheme:
- Use a colorimeter to measure the absorbance of light passing through the milk suspension. (1 mark)
- Lower absorbance indicates increased clarity as protein is digested. (1 mark)
- Take regular readings at fixed time intervals (e.g., every 30 seconds). (1 mark)
- Plot a graph of clarity (absorbance or percentage transmission) against time to observe the rate. (1 mark)
- The steeper the slope, the faster the rate of trypsin activity. (1 mark)
Question 7
Explain why buffers are necessary in experiments investigating enzyme activity at different pH levels. (3 marks)
Mark Scheme:
- Buffers maintain a constant pH, preventing fluctuations caused by the reaction. (1 mark)
- This ensures that any changes in enzyme activity are due to the initial pH conditions, not ongoing pH changes. (1 mark)
- Buffers allow accurate determination of the enzyme’s optimum pH by isolating pH as the only variable. (1 mark)
Question 8
What conclusions can be drawn from a graph of reaction rate vs. pH for trypsin? (5 marks)
Mark Scheme:
- The peak of the graph indicates the optimum pH for trypsin, where the reaction rate is highest. (1 mark)
- On either side of the optimum, the reaction rate declines due to enzyme activity reduction. (1 mark)
- At low pH, trypsin activity decreases as acidic conditions disrupt ionic bonds. (1 mark)
- At high pH, activity also decreases due to alkaline denaturation of the enzyme. (1 mark)
- This pattern reflects the sensitivity of trypsin to charged ion concentrations at varying pH levels. (1 mark)
Question 9
Explain how hydrogen ions (H⁺) affect enzyme structure and activity. (4 marks)
Mark Scheme:
- Hydrogen ions interact with charged R groups in the amino acids of the enzyme. (1 mark)
- These interactions disrupt ionic bonds, altering the enzyme’s 3D shape. (1 mark)
- The change in shape affects the active site, reducing substrate binding and lowering reaction rate. (1 mark)
- Extreme H⁺ concentrations can denature the enzyme, leading to complete loss of activity. (1 mark)
Question 10
Why is pH important in the function of proteases used in biological washing powders? (5 marks)
Mark Scheme:
- Proteases in washing powders are designed to work at specific pH levels. (1 mark)
- The washing powder environment must match the protease’s optimum pH for maximum activity. (1 mark)
- Neutral to slightly alkaline proteases are effective at breaking down protein stains (e.g., blood). (1 mark)
- The use of buffers in washing powders helps maintain the ideal pH for enzyme function. (1 mark)
- Protease stability in these conditions ensures effective stain removal during washing. (1 mark)