1Denaturation: when the shape goes

A protein's shape is held by weak bonds between R groups: hydrogen bonds, and attractions between oppositely charged groups. Denaturation is the loss of that shape. The amino acid sequence is still there; the folding is not. Three things cause it:

• Heat. Higher temperature makes the enzyme's own atoms vibrate harder. Past a point, the hydrogen bonds holding the folds break, and the active site loses its shape.
• pH. pH is a measure of hydrogen ion concentration. Adding or removing H⁺ changes the charges on R groups: a group that was negative may gain an H⁺ and become neutral. That breaks ionic attractions and hydrogen bonds, and it changes the charges in the active site that the substrate needs.
• Chemicals. High salt concentrations, some metal ions and some solvents can also disrupt the bonds that hold the shape.

Is denaturation permanent? Sometimes. If the change was small or brief, some enzymes refold when normal conditions return and regain their activity: their denaturation is reversible. Severe heat usually is not reversible, because the unfolded chains tangle and stick to each other. The only way to know for a given enzyme is to test it: expose it to the condition, return it to its optimum, and measure the rate again.

2Temperature and pH: the optimum

Temperature, Figure 1(a). Read the curve in two parts.

• Rising side. As temperature rises, enzyme and substrate molecules move faster. They collide more often, and more collisions have enough energy to react, so the rate increases. The enzyme's shape is still intact.
• Falling side. Above the optimum temperature, the bonds holding the enzyme's shape start to break. More and more enzyme molecules denature, so even though collisions are still frequent, fewer of them lead to a reaction. The rate falls steeply.

The curve rises gradually and falls fast. Cold slows an enzyme by making collisions fewer, not by denaturing it.

pH, Figure 1(b). Each enzyme has an optimum pH, and moving away from it in either direction lowers the rate, because changing H⁺ concentration changes the charges on R groups. The figure shows two digestive enzymes: pepsin works in the stomach, near pH 2, and trypsin in the small intestine, near pH 8. Each enzyme's optimum suits the place where it works.

Worked example: reading a temperature experiment. Invented data: the initial rate of an enzyme at six temperatures.

The percentage increase in rate from 30 °C to 40 °C:

The optimum lies somewhere between 40 °C and 50 °C, close to 40 °C; the data cannot say exactly where, because no temperature between them was tested. To find it more precisely, test more temperatures in that range.

To test whether the drop at 60 °C is reversible, a student holds the enzyme at 60 °C for ten minutes, cools it to 40 °C and measures again. A rate of 0.4 µmol min⁻¹ against 18.5 before would show the denaturation was not reversed. A rate back near 18.5 would show it was.

Above the optimum, rate falls because the enzyme's shape is lost, not because molecules slow down.

3Substrate and product concentrations

The relative amounts of substrate and product around an enzyme decide how efficiently it works.

More substrate. With few substrate molecules, most active sites are empty at any moment. Adding substrate means more collisions, so the rate rises. At high concentrations, nearly every active site is occupied all the time. The enzyme is now working as fast as it can, and adding more substrate barely changes the rate. To go faster, you would need more enzyme.

More product. Many enzyme-catalysed reactions can run in either direction. If product builds up, the reverse reaction becomes more frequent and the net rate of forward reaction falls. A cell avoids this by using products as soon as they form: in a metabolic pathway, the next enzyme takes each product as its substrate (Topic 3.3). Keeping product low keeps the forward reaction going.

So if a cell stops using the product of enzyme X, predict that X slows down as product accumulates.

4Inhibitors

An inhibitor is a molecule that binds an enzyme and lowers its activity. The two kinds in this course differ in where they bind.

• Competitive inhibitor, Figure 2(a). It is similar in shape and charge to the substrate, and it binds reversibly to the active site. While it is there, the substrate cannot bind. The inhibitor and the substrate compete for the same site, so the outcome depends on which is more common. Add a lot of substrate, and substrate molecules win most of the collisions: the rate climbs back towards the uninhibited maximum.
• Noncompetitive inhibitor, Figure 2(b). It binds an allosteric site, a site on the enzyme other than the active site. Binding changes the enzyme's shape, including the active site, so the enzyme works less well or not at all. Since the inhibitor is not competing for the active site, adding more substrate does not get rid of it. The maximum rate stays lower however much substrate you add.

Worked example: telling them apart from data. Invented initial rates (µmol min⁻¹) for an enzyme, with no inhibitor and with each of two inhibitors at a fixed concentration.

• Claim: X is competitive, Y is noncompetitive.
• Evidence: at high substrate (20 mmol L⁻¹), X's rate (40) almost matches the uninhibited rate (42). Y's rate stays at half the uninhibited rate at every concentration: 5 of 10, 15 of 30, 21 of 42.
• Reasoning: extra substrate can outcompete an inhibitor only if they compete for the same active site. An inhibitor bound elsewhere keeps the same share of enzymes changed in shape, whatever the substrate concentration.

Cells use allosteric binding to regulate their own pathways. In feedback inhibition, the end product of a pathway binds an allosteric site on an enzyme early in the pathway and slows it, so the cell stops making the product when it has enough. You meet this again in Unit 4.