Amylase, pH and Copper Ions: Reading Inhibition Data
A student investigates salivary amylase by mixing 1 cm3 of diluted saliva with 5 cm3 of 1% starch solution and testing a drop with iodine every 30 s until no blue-black colour appears. At pH 7, the times are: 20°C, 8.0 min; 30°C, 4.0 min; 40°C, 2.0 min; 50°C, 3.0 min; 60°C, 10.0 min; 70°C, no colour loss within 15 min. At 40°C, the times are: pH 4, 12.0 min; pH 5, 5.0 min; pH 6, 2.5 min; pH 7, 2.0 min; pH 8, 4.0 min. When 0.1 M CuSO4 is added at 40°C and pH 7, the time is 14.0 min; doubling the starch concentration gives 13.0 min. If the enzyme is first held at pH 4 for 10 min and then returned to pH 7, the time is 2.1 min; if held at 70°C for 10 min and cooled to 40°C, no colour loss occurs in 15 min. The student claims that low pH denatures amylase and that CuSO4 is a competitive inhibitor.
1. Using the times, identify the optimum temperature and pH. Explain the shape of the temperature curve, including the result at 70°C.
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Rate is proportional to 1 ÷ time. At pH 7 the relative rates are: 20°C, 0.125; 30°C, 0.25; 40°C, 0.50; 50°C, 0.33; 60°C, 0.10; 70°C, 0. The optimum is 40°C. From 20°C to 40°C, higher temperature gives more kinetic energy and more frequent enzyme–substrate collisions, so rate rises. Above 40°C, non-covalent bonds holding the active site begin to break. At 50°C activity is already falling, at 60°C it is much lower, and at 70°C the enzyme is denatured. The pre-incubation result confirms this: enzyme held at 70°C and then cooled to 40°C does not recover activity, which is the signature of irreversible denaturation rather than simple slowing.
At 40°C the pH rates are: pH 4, 0.083; pH 5, 0.20; pH 6, 0.40; pH 7, 0.50; pH 8, 0.25. The optimum is pH 7, with pH 6 close. pH extremes change the ionisation of amino-acid side chains in the active site and of the substrate, reducing binding. The pH effect is not automatically denaturation, as the recovery experiment shows.
2. Is CuSO4 a competitive inhibitor? Use the doubled-substrate result and explain your reasoning.
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A competitive inhibitor binds the active site, so raising the substrate concentration can outcompete it and restore the rate. Here, doubling the starch concentration changed the time only from 14.0 min to 13.0 min. The relative rates are 1 ÷ 14 = 0.071 and 1 ÷ 13 = 0.077, compared with 1 ÷ 2.0 = 0.50 without CuSO4. Doubling substrate produced only a negligible recovery, so the inhibition is not competitive.
The result is consistent with non-competitive inhibition or with irreversible inactivation of the enzyme by Cu2+. Copper ions can bind outside the active site, alter the protein's shape, or cause precipitation/denaturation. To distinguish these possibilities, the enzyme could be pre-incubated with CuSO4, then Cu2+ removed by dialysis before assay; recovery would argue against irreversible inactivation. A Lineweaver–Burk plot would also separate competitive from non-competitive kinetics. On the evidence given, the student's claim is not supported.
3. A student says low pH denatures amylase. Evaluate this claim using the recovery experiment.
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Denaturation is a loss of the enzyme's three-dimensional shape, usually irreversible. If pH 4 had denatured amylase, returning the enzyme to pH 7 would not restore activity. But enzyme held at pH 4 for 10 min and then returned to pH 7 gave a time of 2.1 min, almost the same as the 2.0 min control. That is essentially full recovery. Low pH therefore reversibly inhibits amylase, probably by changing the ionisation of groups at the active site, rather than denaturing it.
The contrast with temperature is important. Enzyme held at 70°C and cooled showed no activity in 15 min, which is consistent with irreversible denaturation. The pH 4 treatment may still cause some slow denaturation if held much longer, but the recovery data argue against substantial irreversible loss under these conditions. The student's claim is therefore too strong: low pH reduces activity, but it does not necessarily denature the enzyme.