Enzymes: Question 4
Syllabus 3.2
A research group studies an enzyme, enzyme X, and two molecules, inhibitor P and inhibitor Q, each of which reduces the rate at which enzyme X converts its substrate into product. The group measures the initial rate of reaction across a range of substrate concentrations, with no inhibitor present, with inhibitor P present, and with inhibitor Q present. The concentration of enzyme X and of each inhibitor is kept the same throughout.
| Substrate concentration / mmol dm-3 | 1 | 2 | 5 | 10 | 20 | 50 |
|---|---|---|---|---|---|---|
| Rate, no inhibitor / arbitrary units | 10 | 18 | 30 | 38 | 42 | 44 |
| Rate, with inhibitor P / arbitrary units | 3 | 6 | 14 | 24 | 36 | 43 |
| Rate, with inhibitor Q / arbitrary units | 5 | 9 | 15 | 19 | 21 | 22 |
(a) Using the data in the table, state, with a reason, which inhibitor is a competitive inhibitor and which is a non-competitive inhibitor. [3]
(b) Explain, in terms of where each type of inhibitor binds on the enzyme, why the effect of inhibitor P becomes much smaller at high substrate concentration, but the effect of inhibitor Q does not. [4]
(c) State whether the Km of enzyme X is increased, decreased or unchanged in the presence of (i) inhibitor P and (ii) inhibitor Q, and explain what this indicates about the substrate concentration needed to reach half of the maximum rate in each case. [3]
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Worked solution
Part (a): Identifying the competitive and non-competitive inhibitor
The key clue is what happens at the highest substrate concentration tested (50 mmol dm-3):
- With inhibitor P, the rate reaches 43, almost exactly the same as the uninhibited rate of 44.
- With inhibitor Q, the rate reaches only 22, around half of the uninhibited rate, and shows no sign of approaching 44 even at this high substrate concentration.
An inhibitor whose effect disappears at high substrate concentration is a competitive inhibitor, because there is now so much substrate available that it out-competes the inhibitor for the enzyme’s active sites. An inhibitor whose effect persists even at high substrate concentration is a non-competitive inhibitor, because adding more substrate cannot remove it from wherever it is bound.
So inhibitor P is competitive and inhibitor Q is non-competitive.
Part (b): Why substrate concentration overcomes P but not Q
Inhibitor P (competitive): a competitive inhibitor has a molecular shape similar enough to the substrate that it can bind reversibly to the enzyme’s active site, directly blocking the substrate from binding there. At low substrate concentration, inhibitor molecules occupy a significant fraction of active sites, greatly reducing the rate. But as substrate concentration rises, substrate molecules become far more numerous than inhibitor molecules, so at any given moment substrate is much more likely to bind a free active site than the inhibitor is. Substrate effectively out-competes the inhibitor for access to the active site, so the rate climbs back up towards the uninhibited rate.
Inhibitor Q (non-competitive): a non-competitive inhibitor does not resemble the substrate and does not bind at the active site at all (it binds at a separate site elsewhere on the enzyme. This binding changes the enzyme’s overall three-dimensional (tertiary) structure, which distorts the shape of the active site itself, so the enzyme cannot bind substrate properly (or cannot catalyse the reaction even if substrate does bind) at any active site affected in this way. Because inhibitor Q is not competing with substrate for the same site, adding more substrate does nothing to displace it) the substrate and the inhibitor are not competing for the same location, so the inhibitor’s effect on the enzyme molecules it is bound to cannot be “diluted out” by extra substrate.
Part (c): Effect on Km
(i) Inhibitor P (competitive) (Km increased. Because more substrate is now needed to out-compete the inhibitor and occupy enough active sites to reach half of the maximum rate, the substrate concentration required to reach half-Vmax is higher than without the inhibitor. Since Km is defined as the substrate concentration giving half of Vmax, this means Km increases) indicating a lower apparent affinity between enzyme and substrate while the competitive inhibitor is present (the enzyme’s true affinity is unchanged, but the inhibitor makes it behave as if affinity were lower).
(ii) Inhibitor Q (non-competitive), Km unchanged. The active sites that are not affected by inhibitor Q bind substrate exactly as effectively as in the uninhibited enzyme, the inhibitor does not change the shape or chemistry of active sites it has not bound near. So the substrate concentration needed to reach half of the maximum rate is the same as before; it is Vmax itself, not Km, that falls because a fraction of the enzyme population is no longer catalytically functional.
Final answers
- (a) P is competitive (its effect is largely overcome at high substrate concentration, 43 vs 44); Q is non-competitive (it plateaus at 22, well below 44, even at the highest substrate concentration).
- (b) P binds at the active site and is out-competed by excess substrate; Q binds elsewhere, distorting the active site indirectly, so extra substrate cannot displace it.
- (c) (i) Km increased with inhibitor P (more substrate needed to reach half-Vmax; lower apparent affinity). (ii) Km unchanged with inhibitor Q (unaffected active sites bind substrate with the same affinity; only Vmax falls).