Catalase inhibition in potato extract: testing a claimed non-competitive inhibitor
A student investigates catalase in potato tissue. 20 g of peeled potato is blended with 30 cm3 of 0.10 mol dm−3 phosphate buffer at pH 7.0 and filtered to make 50 cm3 of extract. For each trial, 5.0 cm3 of extract is mixed with 10.0 cm3 of hydrogen peroxide solution in a sealed tube at 25 °C, and oxygen volume is read from a gas syringe every 10 s for 2 min. The initial rate is taken from the steepest linear section. The experiment is repeated after adding a reversible inhibitor to give 0.50 mmol dm−3 in the extract. At hydrogen peroxide concentrations of 20, 40, 80, 160 and 320 mmol dm−3, control rates are 0.42, 0.68, 0.96, 1.20 and 1.38 cm3 min−1. With inhibitor present, the rates are 0.18, 0.35, 0.62, 0.98 and 1.26 cm3 min−1. The student concludes that the inhibitor is non-competitive “because the rate is always lower”.
1. Calculate the percentage inhibition at 20 and at 320 mmol dm−3 hydrogen peroxide. Use all the data to evaluate the student's conclusion.
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At 20 mmol dm−3: (0.42 − 0.18) ÷ 0.42 × 100 = 57.1% inhibition. At 320 mmol dm−3: (1.38 − 1.26) ÷ 1.38 × 100 = 8.7% inhibition. The degree of inhibition falls from about 57% to about 9% as substrate concentration increases 16-fold. More importantly, the inhibited rate approaches the control rate at high substrate concentration: 1.26 compared with 1.38 cm3 min−1. This means the maximum rate, Vmax, is not substantially reduced; the apparent Km is increased because more substrate is needed to reach half Vmax. That is the signature of competitive inhibition, not non-competitive inhibition. A non-competitive inhibitor would reduce Vmax at every substrate concentration and would not be reversed by raising [S]. The student's conclusion is therefore not supported. The small remaining difference at 320 mmol dm−3 could be experimental variation or a mixed effect, so the claim should be tested with replicates and higher substrate concentrations to confirm the plateau.
2. Explain the mechanism of the inhibition you have identified and why increasing substrate concentration reduces its effect.
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A competitive inhibitor has a shape similar to the substrate and binds to the enzyme's active site. It competes with substrate molecules for the same site, so at any moment fewer enzyme molecules are available to form enzyme–substrate complexes. The rate is lower at a given substrate concentration because the probability of substrate binding is reduced. Raising substrate concentration increases the chance that a substrate molecule, rather than an inhibitor molecule, occupies the active site. At sufficiently high [S], substrate outcompetes the inhibitor, most active sites are occupied by substrate, and the rate approaches the uninhibited Vmax. The inhibitor does not permanently damage the enzyme; its effect is reversible. This differs from non-competitive inhibition, where the inhibitor binds at a different site and lowers Vmax because the enzyme–substrate complex is less able to form product, regardless of substrate concentration.
3. Suggest two improvements to the method that would make the comparison between control and inhibited rates more reliable.
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First, standardise the enzyme preparation: use the same batch of potato extract, the same mass of potato and the same filtration procedure for all trials, because catalase concentration affects rate. Pre-incubate the extract with inhibitor for a fixed time at 25 °C before adding substrate so that binding reaches equilibrium. Second, control temperature with a thermostatically controlled water bath and check pH with a pH meter rather than assuming the buffer is correct; both affect catalase activity. Measure the initial rate over a short period, such as the first 30 s, before substrate depletion or product inhibition becomes significant. Use at least three replicates at each concentration and calculate means; this reduces the influence of anomalous readings. Finally, extend the substrate range above 320 mmol dm−3 to show whether the inhibited and control rates truly converge at a plateau, which is needed to distinguish competitive from non-competitive inhibition confidently.