Questions on enzyme inhibition and metabolic control

Question 1

Compare a competitive inhibitor with a non-competitive inhibitor, referring to where each one binds and what each does to the enzyme.

Accepted Answer

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 2

Explain how end-product inhibition controls the rate of a metabolic pathway, and explain why inhibiting an early committed step prevents substrate and ATP being spent on intermediates the cell does not need.

Accepted Answer

B1 the final product of the pathway acts as a non-competitive inhibitor of the enzyme catalysing the first committed step, binding at an allosteric site B1 as product accumulates more of that 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 a later enzyme instead would leave the earlier steps running, so substrate and ATP would be spent building intermediates the cell has no use for

Question 3

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.

Accepted Answer

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; for continuous large-scale production the savings from reuse outweigh these, so immobilising is the better choice

Question 4

Compare a reversible inhibitor with an irreversible inhibitor, referring to the type of bond formed and to whether activity can be restored.

Accepted Answer

B1 a reversible inhibitor is held by weak interactions, such as hydrogen bonds, ionic bonds and hydrophobic interactions, the same kind of forces that hold a substrate B1 an irreversible inhibitor forms a covalent bond with the enzyme B1 a reversible inhibitor can dissociate, so the enzyme regains activity once the inhibitor's concentration falls, whereas an irreversible inhibitor leaves that enzyme molecule permanently inactive B1 activity lost to an irreversible inhibitor is restored only by the cell synthesising new enzyme molecules, not by removing the inhibitor

Question 5

Explain how a statin lowers the rate of cholesterol synthesis in the liver, and explain why its effect wears off once a patient stops taking it.

Accepted Answer

B1 a statin has a shape similar to the substrate of HMG-CoA reductase, the enzyme controlling the rate-limiting step of cholesterol synthesis B1 it competes with the natural substrate for the active site, so fewer enzyme-substrate complexes form and the rate of that step, and so of the whole pathway, falls B1 a statin is a reversible, competitive inhibitor, held by weak interactions rather than a covalent bond B1 once the drug is stopped its concentration in the liver falls, so it dissociates from the enzyme and normal enzyme activity, and cholesterol synthesis, returns

Question 6

Without inhibitor, an enzyme has Vmax = 60 μmol min⁻¹ and Km = 1.0 mmol dm⁻³. With inhibitor P present, Vmax = 30 and Km = 1.0. With inhibitor Q present, Vmax = 60 and Km = 4.0. Identify the type of each inhibitor, and identify the evidence that rules out the other type in each case.

Accepted Answer

B1 P is a non-competitive inhibitor, because Vmax has fallen, from 60 to 30, while Km is unchanged B1 a competitive inhibitor is ruled out for P because the unchanged Km shows the sites still working bind substrate exactly as before, and raising substrate concentration could not restore the lost Vmax B1 Q is a competitive inhibitor, because Vmax is unchanged, still 60 and reachable given enough substrate, while Km has increased, from 1.0 to 4.0 B1 a non-competitive inhibitor is ruled out for Q because a fixed loss of active sites would lower Vmax however much substrate was added, which is not observed here

Question 7

A new pesticide binds permanently and covalently to an insect enzyme essential for nerve function, at a site away from its active site. Suggest the type of inhibitor this pesticide is, giving your reasoning, and suggest why washing an exposed insect in water would not restore the enzyme's activity.

Accepted Answer

B1 it is a non-competitive inhibitor: it binds away from the active site, so it does not compete with the substrate, and it alters the enzyme's tertiary structure so the active site changes shape B1 the covalent bond it forms makes it irreversible, unlike the weak interactions that hold a reversible inhibitor in place B1 washing removes only what is not chemically bonded to the enzyme, and a covalent bond does not break under those conditions B1 activity in the affected insect would return only once new enzyme molecules were synthesised, not because the existing ones recovered

Question 8

Explain why penicillin kills growing bacteria but does no harm to human cells.

Accepted Answer

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 9

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.

Accepted Answer

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 10

Describe two advantages of immobilising an enzyme for an industrial process, other than the ability to reuse it.

Accepted Answer

B1 the product contains none of the enzyme, because the enzyme stays on its support rather than mixing with the product, so no separation step is needed B1 the support holds the tertiary structure in place, so the immobilised enzyme tolerates higher temperatures than the same enzyme free in solution, allowing a faster, hotter process B1 the process can be run continuously, with substrate pumped through a packed column, rather than in separate batches

Question 11

Name the enzyme immobilised to produce lactose-free milk, name the enzyme used to convert glucose syrup into a sweeter fructose syrup, and name the enzyme used to remove a side chain from natural penicillin.

Accepted Answer

B1 lactase, immobilised on beads B1 glucose isomerase, for fructose syrup B1 penicillin acylase, for semi-synthetics

Question 12

Explain why raising the substrate concentration relieves competitive inhibition but has no effect on non-competitive inhibition.

Accepted Answer

B1 a competitive inhibitor competes with the substrate for the same active site, so raising the substrate concentration increases the chance that a substrate molecule occupies a site before the inhibitor does B1 a non-competitive inhibitor binds at a separate allosteric site, not the active site, so it does not compete with substrate molecules for a place B1 with the active sites of the affected enzyme molecules already distorted, adding more substrate gives them nothing to bind to, so the inhibition is not relieved

Question 13

State the effect a non-competitive inhibitor has on the Vmax of an enzyme, and state its effect on the Km.

Accepted Answer

B1 Vmax is lowered by a non-competitive inhibitor B1 Km is unchanged by a non-competitive inhibitor

Question 14

State the effect a competitive inhibitor has on the Vmax of an enzyme-catalysed reaction, and state its effect on the apparent Km.

Accepted Answer

B1 Vmax is unchanged by a competitive inhibitor, and is still reached given enough substrate B1 the apparent Km is increased, because a higher substrate concentration is needed to reach half of Vmax

Question 15

Phosphofructokinase catalyses an early step in glycolysis, using ATP as one of its substrates. A high concentration of ATP also inhibits the enzyme by binding to a second site, away from the active site, which changes the shape of the active site. State whether ATP acts here as a competitive or a non-competitive inhibitor, and explain how this inhibition matches the rate of glycolysis to the cell's demand for ATP.

Accepted Answer

B1 non-competitive, because it binds at a site other than the active site and changes the active site's shape, so fewer enzyme-substrate complexes form B1 when ATP is plentiful the enzyme is inhibited and glycolysis slows, so glucose is not used to make ATP that is not needed; as ATP is used and its concentration falls, the inhibition is released and glycolysis speeds up again