Enzymes revision, GCSE & A-Level Biology

What are enzymes?

Enzymes are biological catalysts: globular proteins that speed up chemical reactions in living organisms without being used up. They work by lowering the activation energy of a reaction, the minimum energy needed for reactants to collide successfully and form products.

Each enzyme has a specifically shaped active site that is complementary to one substrate, a property called specificity. The substrate binds to the active site to form an enzyme-substrate complex, and the products are released, leaving the enzyme unchanged and ready to catalyse another reaction.

• Biological catalysts: speed up reactions without being consumed.
• Lower activation energy, so reactions proceed faster at body temperature.
• Specific: each enzyme typically catalyses one reaction with one substrate.
• Examples: amylase digests starch to maltose; trypsin digests proteins; RuBisCO fixes CO2 in photosynthesis.

Enzyme structure and the active site

Enzymes are globular proteins with a complex tertiary structure held together by hydrogen bonds, ionic bonds and disulfide bridges. The shape of the active site is determined by the precise folding of the polypeptide chain, which in turn depends on the sequence of amino acids (primary structure).

If the bonds holding the tertiary structure are disrupted, the active site changes shape and the substrate no longer fits. This is denaturation: the enzyme loses its catalytic function. Denaturation can be caused by high temperature or extremes of pH, and it is usually irreversible.

• Active site: a small region of the enzyme, complementary in shape to the substrate.
• Tertiary structure: the 3D folding that creates the active site.
• Denaturation: disruption of tertiary structure, active site changes shape, substrate no longer fits.

Lock and key vs induced fit

At GCSE you learn the lock-and-key model: the substrate has a shape that fits exactly into the rigid active site, like a key in a lock, forming an enzyme-substrate complex.

At A-Level the induced-fit model replaces this. The active site is not rigid; it is flexible. When the substrate binds, the active site changes shape slightly to mould around the substrate. This puts strain on the bonds within the substrate, lowering the activation energy and making the reaction more likely to occur.

The induced-fit model explains how enzymes can catalyse reactions: the distortion of the active site destabilises the substrate, lowering the energy barrier for the reaction to proceed.

• Lock and key (GCSE): rigid active site, substrate fits exactly.
• Induced fit (A-Level): active site flexes around the substrate, straining its bonds.
• Both models depend on the active site being complementary to the substrate.

Factors affecting enzyme activity

Temperature: as temperature rises, molecules have more kinetic energy, so they collide more frequently and with more energy, and the rate increases. Above the optimum temperature, the hydrogen and ionic bonds in the tertiary structure break, the active site changes shape and the enzyme denatures. The rate falls sharply.

pH: each enzyme has an optimum pH. Away from the optimum, changes in the charges on amino acid side chains disrupt the ionic and hydrogen bonds holding the active site in shape. Extremes of pH can denature the enzyme; small shifts reduce activity reversibly.

Substrate concentration: at low substrate concentration, rate increases linearly because there are free active sites. As concentration rises, all active sites become occupied (saturation) and the rate reaches a maximum (Vmax), which cannot be exceeded by adding more substrate.

• Temperature: rate rises to an optimum, then drops sharply as the enzyme denatures.
• pH: each enzyme has an optimum (pepsin pH 2, amylase pH 7, trypsin pH 8).
• Substrate concentration: rate increases until Vmax, when all active sites are saturated.
• Enzyme concentration: if enzyme is limiting, adding more increases the rate proportionally.

Competitive and non-competitive inhibition

Competitive inhibitors have a shape similar to the substrate and bind reversibly to the active site, blocking the substrate. The effect can be overcome by increasing substrate concentration, which outcompetes the inhibitor for the active site. At A-Level, competitive inhibitors raise the apparent Km but do not change Vmax.

Non-competitive inhibitors bind to a site other than the active site (an allosteric site), changing the shape of the active site so the substrate can no longer bind. The effect cannot be overcome by adding more substrate. At A-Level, non-competitive inhibitors lower Vmax but do not change Km.

Named examples: statins competitively inhibit HMG-CoA reductase to lower cholesterol; penicillin non-competitively inhibits a bacterial cell-wall enzyme; cyanide non-competitively inhibits cytochrome oxidase in respiration.

• Competitive: binds active site, overcome by more substrate, raises Km, Vmax unchanged.
• Non-competitive: binds allosteric site, cannot be overcome, lowers Vmax, Km unchanged.
• Reversible vs irreversible: some inhibitors (e.g. cyanide) bind irreversibly.
• Drugs as inhibitors: statins, penicillin, aspirin, ACE inhibitors.

Immobilised enzymes (A-Level)

Immobilised enzymes are enzymes attached to an inert support material or trapped in a gel, so they are held in place while substrate flows past. This allows continuous processes, easy recovery and reuse of the enzyme, and greater stability at extremes of temperature and pH.

Common applications include lactase immobilised on beads to produce lactose-free milk, and glucose isomerase to convert glucose to fructose in the production of high-fructose corn syrup. A-Level questions may ask you to evaluate the advantages of immobilisation over using free enzymes in solution.

• Advantages: reusable, continuous operation, more stable, easy product separation.
• Disadvantages: may reduce enzyme activity slightly, higher setup cost.
• Example: lactase beads for lactose-free milk.