Enzymes

📚The biology

What an enzyme does

Enzymes are globular proteins that act as biological catalysts. Some work inside the cell that made them (intracellular — catalase, the respiratory enzymes); some are secreted to act outside it (extracellular — amylase, protease).

An enzyme has an active site, a depression whose shape and charge distribution are complementary to one particular substrate. Substrate binds to form an enzyme–substrate complex; the reaction occurs; product is released and the enzyme is unchanged. The catalytic effect comes from lowering the activation energy — the energy barrier the reaction must cross — so a far greater proportion of collisions at a given temperature are successful.

Lock-and-key and induced fit

The lock-and-key hypothesis treats the active site as a rigid shape that only the correct substrate fits, which explains specificity simply. The induced-fit hypothesis is the modern refinement: the active site is flexible, and binding of the substrate causes the site to change shape slightly so that it moulds around the substrate. That conformational change strains the bonds in the substrate, which is a better account of how the activation energy falls, and it explains why some enzymes accept a small range of related substrates.

Both models explain specificity; only induced fit explains the mechanism. Say both if a question asks you to compare.

Following the rate of a reaction

Two directions, both examinable:

• Product formation — catalase. Catalase breaks hydrogen peroxide into water and oxygen. Collect the oxygen in a gas syringe or by displacement and record volume against time.
• Substrate disappearance — amylase. Amylase digests starch. Remove a drop at fixed intervals into iodine solution on a spotting tile; the blue-black fades and eventually the iodine stays orange-brown, at which point no starch remains.

A colorimeter converts a colour change into a numerical absorbance, removing the subjectivity of judging colour by eye and letting you plot a proper graph.

Whichever method, the curve of product against time is steep at first and flattens. Always take the initial rate, the gradient of the tangent at time zero, because that is the only point at which substrate concentration is the value you set. Later the substrate is being used up and the rate falls for a reason that has nothing to do with the variable you are testing.

The five factors

Temperature. Rate rises with temperature because molecules have more kinetic energy, so there are more frequent collisions with sufficient energy, more enzyme–substrate complexes per second. Above the optimum, increasing vibration breaks the hydrogen and ionic bonds holding the tertiary structure; the active site changes shape, is no longer complementary, and the enzyme is denatured. The fall is steep and, unlike the rise, irreversible.

pH. A narrow optimum. Away from it, excess H⁺ or OH⁻ ions interfere with the ionic bonds and hydrogen bonds of the tertiary structure and with the charges on R groups in the active site, so substrate no longer binds. Small deviations are reversible; large ones denature. This is why pH must be held with a buffer solution, not by adding acid at the start and hoping.

Enzyme concentration. With substrate in excess, rate is directly proportional to enzyme concentration — more active sites, more complexes per second. The line only levels off if substrate becomes limiting.

Substrate concentration. Rate rises steeply, then levels off at V_max. At low concentrations the active sites are the limiting factor's opposite — there is spare capacity, so adding substrate adds rate. At high concentrations every active site is occupied as soon as it is free: the enzyme is saturated, and adding more substrate changes nothing.

V_max and K_m

K_m, the Michaelis–Menten constant, is the substrate concentration at which the rate is half of V_max. You read it off the graph: find V_max from the plateau, halve it, go across to the curve, drop down to the x-axis.

The interpretation is the part that gets tested: a low K_m means a high affinity for the substrate, because only a little substrate is needed to reach half maximum rate. A high K_m means low affinity. K_m therefore lets you compare two enzymes acting on the same substrate, or one enzyme's affinity for different substrates.

Inhibitors

• Competitive. A molecule similar in shape to the substrate binds to the active site and blocks it. Substrate and inhibitor compete, so the effect depends on their relative concentrations. Raising substrate concentration far enough out-competes the inhibitor, so V_max is still reached — it just takes more substrate. On the graph: the curve rises more slowly, K_m appears higher, but the plateau is unchanged.
• Non-competitive. The inhibitor binds elsewhere — at an allosteric site — and changes the shape of the enzyme, so the active site is no longer complementary. Substrate cannot out-compete it because they are not competing for the same place. V_max is reduced and no amount of extra substrate restores it.

Both of these are reversible inhibitors, which is what the syllabus asks for. The distinguishing experiment is always the same: raise the substrate concentration and see whether the original V_max comes back.

Immobilised enzymes

Enzymes can be trapped in beads of sodium alginate, made by dripping an enzyme–alginate mixture into calcium chloride solution. Compared with free enzyme in solution, an immobilised enzyme usually has a slightly lower rate, because the substrate must diffuse into the bead to reach the active sites. That cost buys real advantages:

• the enzyme is easily recovered and reused, so it is cheaper;
• the product is not contaminated with enzyme, so downstream purification is simpler — important for food and medical products;
• the enzyme is more stable to changes in temperature and pH, because the matrix supports its tertiary structure;
• the reaction can be run continuously by passing substrate through a column of beads.