Biology › Enzymes and metabolic control › Inhibition: how cells, poisons and medicines all turn enzymes down
Inhibition: how cells, poisons and medicines all turn enzymes down
Two molecules can slow the same enzyme by completely different routes, and a graph will tell you which is which. The same two mechanisms explain how a bacterium is killed by penicillin, how a statin lowers cholesterol, and how a cell stops making something it already has enough of.
Before this The active site and induced fit · Substrate concentration and saturation
Before you start
An inhibitor must be a poison, and a cell would never make one on purpose. Cyanide and heavy metals get the headlines, so inhibition sounds like something that happens to a cell rather than something a cell does. In fact inhibition is the main way metabolism is regulated at all. Your cells are inhibiting their own enzymes at this moment, deliberately and reversibly, because the alternative is manufacturing molecules they already have in surplus. The molecule doing most of that inhibiting right now is ATP.
What you should be able to do
- Distinguish competitive from non-competitive inhibition by where the inhibitor binds and what it does to the active site.
- State the effect of each type on Vmax and on Km, and explain both effects in words rather than by quoting the graph.
- Explain why raising the substrate concentration relieves one kind of inhibition and not the other.
- Describe end-product inhibition and say why acting on the first enzyme of a pathway is the efficient choice.
- Give the advantages of immobilising an enzyme for an industrial process, and one disadvantage.
Two ways to get in the way
An inhibitor is any molecule that reduces the rate of an enzyme-catalysed reaction. Two mechanisms matter at A level, and the whole comparison turns on one question: where does the inhibitor bind?
A competitive inhibitor has a shape similar enough to the substrate's that it fits the active site. While it is in there the substrate cannot be, so fewer enzyme-substrate complexes form and the rate falls. Nothing is damaged: the two molecules take turns, and which wins a given site depends on their relative concentrations.
A non-competitive inhibitor binds somewhere else entirely, at what is often called the allosteric site. Binding there changes the tertiary structure of the whole protein, and the active site — a product of that structure — changes shape with it, so the substrate is no longer complementary. The two molecules were never competing for the same place, so flooding the mixture with substrate does not help at all.
- Competitive inhibitor
- A molecule with a shape similar to the substrate that binds to the active site, preventing the substrate from binding.
- Non-competitive inhibitor
- A molecule that binds to a site other than the active site, altering the enzyme's tertiary structure so that the active site is no longer complementary to the substrate.
- Allosteric site
- A binding site on an enzyme away from the active site, at which a regulatory molecule binds.
Saying what the graphs show, in words that score
Both types lower the rate, so a single measurement cannot tell them apart. Vary the substrate concentration and they separate at once.
With a competitive inhibitor present, the rate at any given substrate concentration is lower, but the curve is still climbing towards the same maximum. Add enough substrate and it wins the great majority of collisions with active sites, the inhibitor is outcompeted, and Vmax is reached after all. What changed is how much substrate that takes, so Km — the substrate concentration giving half the maximum rate — appears larger. Say it as 'Vmax is unchanged and still reachable, but a higher substrate concentration is needed, so the apparent Km increases'.
With a non-competitive inhibitor present, a fixed proportion of the enzyme molecules are out of action however much substrate you add, so the maximum rate itself is lower: Vmax falls. The molecules left unaffected have perfectly normal active sites and bind substrate exactly as they always did, so the concentration needed to reach half of the new maximum is what it always was: Km is unchanged. That second half is the sentence candidates miss.
| Competitive | Non-competitive | |
|---|---|---|
| Where it binds | The active site | A site elsewhere on the enzyme |
| Shape | Similar to the substrate | Unrelated to the substrate |
| Effect on the active site | Blocks it | Changes its shape |
| Effect of adding more substrate | Inhibition is overcome | No effect |
| Vmax | Unchanged, still reachable | Lowered |
| Km | Increased (apparent) | Unchanged |
Identifying an inhibitor from three numbers
Without inhibitor, an enzyme has Vmax = 80 μmol min⁻¹ and Km = 0.5 mmol dm⁻³. With inhibitor X, Vmax = 80 and Km = 2.0. With inhibitor Y, Vmax = 44 and Km = 0.5. Identify each inhibitor and justify your answer.
Take X first. Vmax is unchanged, so given enough substrate the enzyme still reaches full speed. Km has quadrupled, so it takes four times as much substrate to get halfway there. Both fit a molecule occupying the active site part of the time and being displaced by substrate: X is competitive.
Y is the mirror image. Km is untouched, so the sites that are working bind substrate exactly as they always did; Vmax has fallen by 45%, so some of the enzyme is out of action no matter how much substrate is supplied. That is non-competitive.
The wording to avoid is 'Y binds more strongly'. Location separates the two, not strength: Y could be held weakly and reversibly and still lower Vmax, because the sites it distorts cannot be rescued by substrate.
Reversible, irreversible, and the medicines that use each
Cut the same set another way: some inhibitors let go, and some do not.
Reversible inhibitors are held by hydrogen bonds, ionic bonds and hydrophobic interactions — the same weak forces that hold a substrate. They come off again and the enzyme recovers. Most competitive inhibitors work this way, and so does essentially all deliberate metabolic regulation, since a cell needs to be able to change its mind.
Irreversible inhibitors form a covalent bond with the enzyme. That molecule is finished, and the only recovery available is synthesising a fresh one. Mercury and silver ions behave this way, binding the sulfur in cysteine side chains, so the disulfide bridges those cysteines should have formed never do and the fold collapses.
Penicillin is the example worth knowing properly. Bacteria cross-link the strands of their peptidoglycan wall using a transpeptidase, and penicillin resembles the piece of peptidoglycan that enzyme normally grips closely enough to enter the active site — where it then reacts, forming a covalent bond that never breaks. The bacterium keeps growing, cannot strengthen its wall, and bursts as water enters by osmosis. The same fact explains why penicillin does nothing to you: no peptidoglycan, no transpeptidase.
Statins are the reversible counterpart: competitive inhibitors of HMG-CoA reductase, the enzyme controlling the rate-limiting step of cholesterol synthesis in the liver, which work because they look enough like its substrate to occupy the site. That is a good property in a drug taken daily — stop taking it and the enzyme goes back to normal.
Switching a pathway off at the top
Cellular products are rarely made in one step. Glucose becomes pyruvate in ten, each with its own enzyme, and dozens of pathways like it run at once inside a cell that has no supervisor. What stops one overproducing?
The answer is end-product inhibition. The final product of the pathway acts as a non-competitive inhibitor of the first enzyme in the sequence, binding at an allosteric site. As product accumulates, more of that first enzyme is inhibited and the pathway slows; as product is used up, the inhibitor comes off and the pathway speeds up again. A thermostat built from one protein and one molecule.
Why the first enzyme rather than the last? Inhibiting the last one leaves the earlier steps running, so the cell fills with intermediates it has no use for and has already spent substrate and ATP making them. Shutting the tap at the top wastes nothing.
The named example to have ready is ATP acting on phosphofructokinase in glycolysis. Phosphofructokinase catalyses an early committing step, and ATP — the pathway's eventual product — binds it at an allosteric site and inhibits it. A cell with plenty of ATP slows its own glucose breakdown; a cell running short releases the brake within seconds. ATP is also a substrate at that enzyme's active site, which is a neat demonstration that the two sites really are different places.
- End-product inhibition
- The inhibition of an enzyme early in a metabolic pathway by the final product of that pathway, so that the rate of the pathway matches demand.
Enzymes stuck down: why industry does it
An enzyme dissolved in its own product creates two problems. It has to be separated out before the product can be sold, and it leaves with the batch, so the next batch needs fresh enzyme. Enzymes are expensive.
Immobilising the enzyme solves both. It is attached to something that does not move: adsorbed onto a clay or resin, covalently bonded to a support such as cellulose, trapped in a gel — alginate beads are the school version — or held behind a partially permeable membrane while substrate diffuses through to it.
| Advantage | Why it follows |
|---|---|
| Recovered and reused | It stays on the support as product flows past |
| Product free of enzyme | No separation step, so lower cost and no protein in the product |
| More stable to heat and pH | The support holds the tertiary structure, so a hotter, faster process is possible |
| Runs continuously | Substrate is pumped through a packed column instead of mixed in batches |
| Easy to stop | Stop the flow and the two are no longer in contact |
The cost is a loss of activity. Fixing an enzyme to a support can block part of the active site or hold it slightly strained, and the substrate has to diffuse to a site that can no longer come to it, so an immobilised enzyme usually works more slowly than the same enzyme free in solution. Set-up costs are higher too. Reuse pays for both within days.
Quote a real example. Lactase immobilised on beads makes lactose-free milk, the milk passing through a column and the enzyme never entering the carton. Glucose isomerase converts glucose syrup into the sweeter fructose syrup used across the food industry. And penicillin acylase strips a side chain from natural penicillin so that semi-synthetic antibiotics can be built on what remains — an immobilised enzyme making the drug from two sections ago.
TRY IT — Choosing a temperature for a column
A dairy passes milk through a column of immobilised lactase. The free enzyme has an optimum of 48 °C but denatures noticeably within an hour at it. Immobilised, it runs at 50 °C for weeks. Suggest why immobilisation makes the higher temperature possible, and give one reason the dairy might still run the column cooler.
Check your answer
Immobilisation restricts how much the polypeptide can move. Attachment to the support, or entrapment in a gel, holds parts of the chain in place, so the vibration that would otherwise break hydrogen and ionic bonds is less able to unfold the tertiary structure. The active site keeps its shape at a temperature that would have destroyed the free enzyme, and rate rises with temperature, so the dairy gets more product per hour as well.
Reasons to run cooler are practical rather than enzymic: warm milk supports bacterial growth, heating costs energy, and prolonged warming affects taste. The first half of this question is about protein structure and the second is about everything else in the factory.
In the exam
- Location, not strength. A competitive inhibitor binds the active site; a non-competitive one binds elsewhere and changes the active site's shape. Answers that say the non-competitive inhibitor 'binds more tightly' have described the wrong difference.
- Learn the Vmax and Km pair as two sentences rather than a table you have to reconstruct: competitive — Vmax unchanged and still reachable, apparent Km higher; non-competitive — Vmax lower, Km unchanged.
- 'Adding more substrate' is the standard discriminating question. It relieves competitive inhibition and does nothing whatever for non-competitive inhibition. Say why, not just which.
- For end-product inhibition the marks are usually two: the product acts on the first enzyme, and it binds somewhere that is not the active site. Add that it prevents waste of substrate and ATP if the question wants an advantage.
- Immobilised-enzyme questions want advantages tied to the industry: reuse, no contamination of the product, continuous operation, higher working temperature. Offer the loss of activity as the disadvantage on an 'evaluate'.
Check yourself
A company has two candidate molecules that both slow the same enzyme in cultured cells. When the substrate concentration in the culture is raised tenfold, molecule A stops having any measurable effect while molecule B works exactly as well as before. Identify each type of inhibition, and advise the company on which molecule is the better starting point for a drug that must work in a tissue where substrate concentration rises and falls sharply after meals.
Answer
Molecule A is a competitive inhibitor. Its effect disappears at high substrate concentration because it binds the active site and is displaced once substrate molecules greatly outnumber it, so Vmax is reached in spite of the inhibitor being present.
Molecule B is a non-competitive inhibitor. It binds away from the active site and alters the enzyme's tertiary structure, so the site is no longer complementary to the substrate. Substrate cannot compete for a place it does not occupy, and the maximum rate is lower however much is available.
For the tissue described, B is the better starting point: a drug based on A would lose its effect exactly when substrate concentration peaked after a meal, so the dose would in effect vary with what the patient had eaten. The advice needs a caveat, though. Non-competitive inhibition cannot be reversed by raising the substrate concentration, so an overdose is harder to counteract, and a molecule that binds one allosteric site may bind others. Check B's specificity across related enzymes, and confirm its binding is reversible, before committing.
Questions
Question 14 marks
Compare a competitive inhibitor with a non-competitive inhibitor, referring to where each one binds and what each does to the enzyme.
Mark scheme
- B1 a competitive inhibitor binds to the active site, whereas a non-competitive inhibitor binds elsewhere, at an allosteric site
- B1 a competitive inhibitor has a shape similar to the substrate, whereas a non-competitive inhibitor has a shape unrelated to it
- B1 a competitive inhibitor blocks the site, whereas a non-competitive inhibitor alters the tertiary structure so the site is no longer complementary to the substrate
- B1 raising the substrate concentration overcomes competitive inhibition, but has no effect at all on non-competitive inhibition
Question 24 marks
Explain how end-product inhibition controls the rate of a metabolic pathway, and explain why it is the first enzyme of the pathway that is inhibited.
Mark scheme
- B1 the final product of the pathway acts as a non-competitive inhibitor of the first enzyme, binding at an allosteric site
- B1 as product accumulates more of that first enzyme is inhibited, so the pathway slows
- B1 as product is used up the inhibitor comes off again, so the pathway speeds up and its rate matches demand
- B1 inhibiting the last enzyme instead would leave the earlier steps running, so substrate and ATP would be spent building intermediates the cell has no use for
Question 34 marks
A dairy producing lactose-free milk must decide whether to immobilise its lactase on a column or to add the free enzyme to each batch. Evaluate the case for immobilising it.
Mark scheme
- B1 the immobilised enzyme stays on its support as the milk flows past, so it is recovered and reused instead of being lost with every batch
- B1 no enzyme leaves with the product, so no separation step is needed and the milk contains none of the enzyme protein
- B1 the support holds the tertiary structure, so the column tolerates a higher temperature and can be run continuously rather than in batches
- B1 against this, an immobilised enzyme works more slowly because part of the active site may be blocked and substrate must diffuse to it, and set-up costs are higher; since reuse repays those costs within days, immobilising is the better choice
Question 43 marks
Explain why penicillin kills growing bacteria but does no harm to human cells.
Mark scheme
- B1 penicillin resembles the piece of peptidoglycan that the bacterial transpeptidase normally grips, closely enough to enter that enzyme's active site
- B1 it then forms a covalent bond with the enzyme, so the inhibition is irreversible, the growing bacterium cannot cross-link its wall, and it bursts as water enters by osmosis
- B1 human cells have no peptidoglycan wall and therefore no transpeptidase, so there is nothing for penicillin to inhibit
Question 53 marks
Mercury ions inhibit many enzymes, and washing the mercury away does not restore activity. Suggest how mercury ions inhibit an enzyme, and suggest why the effect cannot be reversed.
Mark scheme
- B1 mercury ions bind to the sulfur in cysteine side chains
- B1 the disulfide bridges those cysteines would have formed cannot form, so the tertiary structure collapses and the active site loses its shape
- B1 the bond the mercury forms is covalent, so it does not come off, and the only recovery available is to synthesise a fresh enzyme molecule
Question 62 marks
State the effect a non-competitive inhibitor has on the Vmax of an enzyme, and state its effect on the Km.
Mark scheme
- B1 Vmax is lowered by a non-competitive inhibitor
- B1 Km is unchanged by a non-competitive inhibitor
Worth remembering
- Competitive inhibitors bind the active site; non-competitive inhibitors bind elsewhere and change its shape.
- More substrate overcomes competitive inhibition and does nothing for non-competitive inhibition.
- Competitive: Vmax unchanged, apparent Km raised. Non-competitive: Vmax lowered, Km unchanged.
- Reversible inhibitors are held by weak interactions; irreversible ones form a covalent bond, as penicillin does with the transpeptidase that builds a bacterial wall.
- End-product inhibition acts on the first enzyme of a pathway, so no substrate and no ATP is spent making intermediates the cell does not need.