Enzymes: Question 5
Syllabus 5.1
A student is investigating a protease enzyme extracted from a species of bacteria that lives naturally in a soda lake, a body of water with a very alkaline pH of about 10. The student prepares five test tubes, each containing the same volume of buffer solution set to a different pH, adds the same amount of enzyme and the same protein substrate to each tube, and records how long it takes for the protein to be completely broken down in each tube. A shorter time means a faster rate of reaction.
| pH of buffer solution | 4 | 6 | 8 | 10 | 12 |
|---|---|---|---|---|---|
| Time for protein to be completely broken down / min | more than 60 | 45 | 20 | 6 | 30 |
(a) State which pH gives the fastest rate of reaction, and explain how you can tell this from the times recorded. [2]
(b) Explain, in terms of the shape of the enzyme's active site, why hardly any protein is broken down in the test tube at pH 4. [3]
(c) The student did not measure the exact volume of buffer solution added to each test tube, or the exact starting mass of protein substrate used. Suggest two reasons why this makes it difficult to conclude confidently that pH alone is responsible for the different times recorded. [2]
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Worked solution
Part (a): Identifying the fastest rate of reaction
A shorter time to completely break down the protein means the reaction happened faster. The shortest time recorded is 6 minutes, at pH 10, which is much shorter than the times recorded at pH 4, 6, 8 or 12. So pH 10 gives the fastest rate of reaction, and, since this species of bacteria naturally lives in a soda lake of about pH 10, this matches the pH the enzyme would be adapted to work best in.
Part (b): Explaining the effect of pH 4 on the enzyme’s active site
Like temperature, pH affects the shape of an enzyme’s active site. This enzyme’s optimum pH is 10, so pH 4 is a long way from the conditions the enzyme is adapted to.
At a pH this far from the optimum, the bonds holding the enzyme’s normal three-dimensional shape together are disrupted, so the enzyme becomes denatured: the shape of its active site is permanently changed. Because the active site’s shape is no longer complementary to the shape of the protein substrate, the substrate can no longer bind to it, so almost no enzyme-substrate complexes form and hardly any protein is broken down in this test tube.
Part (c): Evaluating the investigation for uncontrolled variables
For a fair test, only pH should differ between the five test tubes. Everything else, including the volume of buffer solution and the starting mass of protein substrate, should be kept the same.
- If the volume of buffer solution was not the same in every tube, the concentration of enzyme or substrate within each tube could differ from one tube to the next, and this alone could speed up or slow down the reaction regardless of pH.
- If the starting mass of protein substrate was not the same in every tube, a tube starting with less substrate could reach “completely broken down” sooner than a tube with more substrate, even if the enzyme in both tubes was working at exactly the same rate.
Because these two variables were not measured and controlled, the student cannot be fully confident that pH alone explains the differences in time recorded between the test tubes.
Final answers
- (a) pH 10 is fastest, shown by the shortest time (6 minutes) to break down the protein.
- (b) pH 4 denatures the enzyme, permanently changing its active site’s shape so the substrate can no longer bind, so hardly any protein is broken down.
- (c) Any two of: uneven buffer volume could change enzyme/substrate concentration; uneven starting substrate mass could make times misleading; without controlling these, pH cannot be confidently identified as the only cause of the different times.