Enzyme inhibition and metabolic control

Competitive and non-competitive inhibition

An inhibitor is any molecule that reduces the rate of an enzyme-catalysed reaction. Two mechanisms are examined at A level, and they are distinguished by where the inhibitor binds.

Describe the drawing

Two enzymes drawn side by side. On the left a competitive inhibitor, similar in shape to the substrate, is sitting in the active site while the substrate waits outside unable to enter. On the right an inhibitor is attached to a site well away from the active site, low on the side of the enzyme, and the active site above has been pulled into a wide shallow shape that the substrate no longer fits.

A competitive inhibitor has a shape similar enough to the substrate's that it fits the active site. While it occupies the site the substrate cannot bind, so fewer enzyme-substrate complexes form and the rate falls. The enzyme is not damaged; the proportion of sites occupied by each molecule 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, being a product of that structure, changes shape with it, so the substrate is no longer complementary. The two molecules do not compete for the same site, so raising the substrate concentration does not relieve the inhibition.

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.

Interpreting the rate curves

Both types lower the rate, so a single measurement does not distinguish them. Varying the substrate concentration does.

Describe the drawing

Rate of reaction plotted against substrate concentration with three curves. The cyan control curve rises and levels off at the maximum rate. The amber competitive curve rises more slowly but climbs towards the same maximum, reaching half of it at a much higher substrate concentration. The coral non-competitive curve levels off against a lower dashed maximum, reaching half of that lower value at the same substrate concentration as the control.

With a competitive inhibitor present, the rate at any given substrate concentration is lower, but the curve still approaches the same maximum. At high substrate concentration, substrate molecules occupy the great majority of active sites, so Vmax is reached. What changes is the substrate concentration required, so Km, the substrate concentration giving half the maximum rate, is apparently larger. State 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 inactive however much substrate is added, so the maximum rate itself is lower: Vmax falls. The unaffected molecules have normal active sites and bind substrate as before, so the concentration needed to reach half of the new maximum is unchanged: Km is unchanged. Include that second statement as well as the first.

That pair describes the ideal, purely non-competitive case examined at A level. Real allosteric and mixed inhibitors often shift Km as well, so treat 'Km unchanged' as the model used in assessment rather than a general rule.

The concentration of the inhibitor is itself a factor. At a fixed substrate concentration, raising the concentration of either type of inhibitor lowers the rate, because at any moment more active sites are occupied by a competitive inhibitor, or more enzyme molecules are distorted by a non-competitive one. The rate falls steeply at first and then more and more gradually towards zero. With a competitive inhibitor the size of the effect depends on how much substrate is competing with it, so the same inhibitor concentration has less effect at a higher substrate concentration; with a non-competitive inhibitor the substrate concentration makes no difference to the fraction of activity lost.

Reversible and irreversible inhibitors

Inhibitors can also be classified by whether the binding is reversible.

Reversible inhibitors are held by hydrogen bonds, ionic bonds and hydrophobic interactions, the same weak forces that hold a substrate. They dissociate and the enzyme recovers activity. Most competitive inhibitors act this way, and so does almost all metabolic regulation within a cell, where the rate must be adjustable in both directions.

Irreversible inhibitors form a covalent bond with the enzyme. That enzyme molecule remains inactive, and activity is restored only by synthesising a replacement. Mercury and silver ions act this way, binding the sulfur of cysteine side chains; that disrupts disulfide bridges and the other interactions holding the tertiary structure, so the fold collapses and takes the active site with it.

Penicillin is the standard named example. Bacteria cross-link the strands of their peptidoglycan wall using a transpeptidase, and penicillin resembles the peptidoglycan fragment that enzyme normally binds closely enough to enter the active site, where it then reacts, forming a covalent bond that does not break. The bacterium continues to grow, cannot strengthen its wall, and bursts as water enters by osmosis. Human cells have no peptidoglycan and no transpeptidase, so penicillin has no equivalent target in them.

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 bind because their shape is similar to that of the substrate. Reversibility is useful in a drug taken daily: when the drug is stopped, enzyme activity returns. Reversibility is not always preferable, however. Penicillin is irreversible and is among the most widely used antibiotics; the requirement depends on the clinical purpose of the drug.

End-product inhibition

Cellular products are rarely made in one step. Glucose is converted to pyruvate in ten reactions, each with its own enzyme, and many such pathways operate simultaneously in one cell. End-product inhibition is the mechanism that matches the rate of a pathway to demand.

Describe the drawing

A four-step pathway drawn left to right: four circles labelled A, B, C and D joined by arrows, with the enzyme for each step named beneath its arrow. A dashed line leaves the end product D, runs down and back along under the pathway, and arrows upwards onto enzyme 1, showing the end product switching off the first committed step of the sequence.

In end-product inhibition, the final product of the pathway acts as a non-competitive inhibitor of the enzyme catalysing the first committed step of the pathway, which is an early regulatory step rather than necessarily the first chemical one. It binds at an allosteric site. As product accumulates, more of that enzyme is inhibited and the pathway slows; as product is used, the inhibitor dissociates and the pathway accelerates again.

Inhibiting the last enzyme instead would leave the earlier steps running, so intermediates would accumulate in the cell after substrate and ATP had been spent making them. The target is the first committed step, the first reaction that serves only this pathway, so earlier steps shared with other pathways continue to operate.

The named example is ATP acting on phosphofructokinase in glycolysis. Phosphofructokinase catalyses an early committed step, and ATP, the eventual product of the pathway, binds it at an allosteric site and inhibits it. A high ATP concentration therefore reduces the rate of glucose breakdown, and a falling ATP concentration relieves the inhibition. ATP is also a substrate at that enzyme's active site, which demonstrates that the two sites are distinct.

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.

Immobilised enzymes in industry

An enzyme dissolved in its own product creates two problems. It must be separated out before the product can be sold, and it leaves with the batch, so the next batch requires 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, of which alginate beads are the school version, or held behind a partially permeable membrane while substrate diffuses through to it.

The disadvantage 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 must diffuse to a site that cannot move towards it, so an immobilised enzyme usually works more slowly than the same enzyme free in solution. Set-up costs are also higher, and both are offset by reuse.

In the school version of this comparison, the enzyme is mixed with sodium alginate solution and dripped into calcium chloride solution, where each drop sets into a bead of calcium alginate with the enzyme trapped inside. The rinsed beads are packed into a syringe barrel and substrate is run through them, while the same volume of substrate is mixed with the same quantity of free enzyme. With lactase and milk, glucose test strips show how quickly product appears in each. The free enzyme usually gives the higher initial rate, but the beads can be rinsed and used again and the product contains no enzyme.

Name a specific 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 removes a side chain from natural penicillin so that semi-synthetic antibiotics can be built on what remains.