Mode of action of enzymes

An enzyme is a globular protein produced by a living organism that acts as a biological catalyst — it increases the rate of a specific biochemical reaction without itself being consumed.

Enzymes share three defining features:

1. They lower activation energy ($E_\text{a}$) — the minimum energy required for colliding particles to react. By providing an alternative pathway via an enzyme-substrate complex, the enzyme reduces $E_\text{a}$ so that more substrate molecules have enough energy to react at any given temperature.
2. They are not used up. Enzymes are regenerated unchanged when products leave the active site, so a single enzyme molecule can catalyse many thousands of reactions per second. A typical turnover number for catalase is around $10^7\,\text{s}^{-1}$.
3. They are specific. Each enzyme catalyses one reaction (or one class of reactions), because its active site is complementary in shape and chemistry to a particular substrate.

Enzymes are essential for life: virtually every cellular reaction — from DNA replication to respiration to digestion — requires an enzyme. Without enzymes, most biochemical reactions would proceed far too slowly to sustain life at body temperature.

Every chemical reaction has an activation energy ($E_\text{a}$) — the minimum energy that reactant particles must possess in order to break existing bonds and form new ones. On an energy profile diagram (energy on the y-axis vs reaction progress on the x-axis), $E_\text{a}$ is the height of the hump between reactants and products.

An enzyme lowers this hump by providing an alternative route via the enzyme-substrate complex. Within the active site, the substrate's bonds are strained and brought into the optimum orientation, so less energy is needed to reach the transition state.

What an enzyme does NOT change:

• The starting energy of the reactants and the final energy of the products are unchanged. The overall energy change ($\Delta H$) is the same with or without the enzyme.
• The position of equilibrium is unchanged. The enzyme speeds up both the forward and the reverse reactions equally, so it only changes how fast equilibrium is reached — not where equilibrium lies.

At a given temperature, the proportion of substrate molecules with energy $\geq E_\text{a}$ is much greater when $E_\text{a}$ is lowered, so the rate of product formation rises sharply.

The catalytic cycle of an enzyme can be summarised as:

$\text{E} + \text{S} \rightleftharpoons \text{ES} \rightarrow \text{EP} \rightarrow \text{E} + \text{P}$

1. Binding. The substrate (S) collides with the enzyme (E) in the correct orientation and binds to the active site, forming an enzyme-substrate complex (ES).
2. Catalysis. Within the ES complex, the active site's R-groups exert strain on the substrate's bonds and may donate or accept protons / electrons. The reaction proceeds, forming an enzyme-product complex (EP).
3. Release. The product no longer fits the active site as snugly (its shape has changed), so it dissociates from the enzyme. The enzyme returns to its original state and is free to bind another substrate.

The active site itself is a small region of the enzyme's surface, usually a cleft or pocket, formed by the tertiary structure of the polypeptide. The R-groups lining the active site come from amino acids that may be far apart in the primary sequence but are brought together by folding.

The lock-and-key model, proposed by Emil Fischer in 1894, was the first widely accepted explanation of enzyme specificity. It assumes:

• The active site has a rigid, pre-formed shape.
• The substrate is exactly complementary to this shape.
• The substrate slots into the active site like a key into a lock, with neither molecule changing shape.
• Only a substrate with the correct shape can bind, which explains specificity.

The lock-and-key model is intuitive and accounts for the basic observation that enzymes are specific. However, it has limitations:

• It cannot easily explain why an enzyme often catalyses reactions on substrates that are slightly different from one another (broad specificity).
• It cannot explain the binding behaviour of competitive inhibitors that only resemble the substrate.
• Experimental evidence (X-ray crystallography from the 1950s onwards) shows that enzymes do change shape on substrate binding.

For these reasons the lock-and-key model has been superseded by the induced-fit model, although you still use the term informally to convey the idea of complementary shapes.

The induced-fit model, proposed by Daniel Koshland in 1958, is the currently accepted explanation of enzyme action. Its key claims are:

• Before binding, the active site is only approximately complementary to the substrate.
• When the substrate enters, the enzyme undergoes a conformational change — the active site moulds itself more tightly around the substrate.
• This shape change strains the bonds within the substrate, distorting it towards the transition state and lowering the activation energy more effectively than a rigid fit could achieve.
• When the products are released, the enzyme returns to its original shape and the cycle repeats.

The induced-fit model explains:

• Why specificity can sometimes be 'broad': a few related substrates can induce slightly different but functional active-site conformations.
• Why competitive inhibitors that only resemble the substrate can bind: they too can induce a partial conformational change, blocking the active site.
• X-ray crystallography evidence: comparing free enzymes with enzymes bound to substrate analogues shows real conformational differences. The classic case is hexokinase, which closes around glucose on binding.

You should be able to compare lock-and-key and induced-fit on three points: rigidity, mechanism of catalysis, and current acceptance.

Each enzyme is specific to a particular substrate (or class of substrates). Specificity arises from two complementary requirements:

1. Shape: the three-dimensional geometry of the active site, determined by the tertiary structure, exactly matches the shape of one substrate.
2. Chemistry: the R-groups lining the active site form specific non-covalent interactions with chemical groups on the substrate — hydrogen bonds (between polar groups), ionic bonds (between charged R-groups and oppositely charged groups on the substrate) and hydrophobic interactions (between non-polar regions).

A substrate that fits the shape but has the wrong chemistry — or vice versa — cannot form a stable ES complex.

Cambridge examiners stress that specificity is not just about shape: recent mark schemes explicitly credit the second mark for mentioning chemistry / R-groups / charge.

Example. The enzyme catalase catalyses only the breakdown of hydrogen peroxide. Other small molecules of similar size — e.g. water, oxygen — cannot react in the active site because they lack the correct chemistry to interact with the haem iron at the catalytic centre.

Enzymes are classified by where they act relative to the cell that produces them.

Intracellular enzymes act inside the cell where they are made. Most enzymes are intracellular — the enzymes of glycolysis, the Krebs cycle, the electron transport chain, DNA replication, transcription and translation are all intracellular. A classic example studied at A Level is:

• Catalase: abundant in liver cells and in peroxisomes of nearly all eukaryotic cells. It catalyses $2\text{H}_2\text{O}_2 \rightarrow 2\text{H}_2\text{O} + \text{O}_2$, detoxifying the hydrogen peroxide produced as a by-product of metabolic oxidation reactions. Catalase has one of the fastest known turnover numbers.

Extracellular enzymes are synthesised in the cell, packaged into vesicles, and secreted across the cell surface membrane (usually by exocytosis). They act outside the cell. Examples:

• Salivary amylase: secreted by the salivary glands into the mouth. It hydrolyses starch to maltose during chewing.
• Pepsin: secreted by gastric chief cells into the stomach lumen. It hydrolyses proteins to shorter peptides at low pH (~2).
• Trypsin: secreted by the pancreas (as inactive trypsinogen) into the small intestine, where it is activated and hydrolyses proteins to shorter peptides at pH ~8.
• Digestive lipases: secreted by the pancreas into the small intestine to hydrolyse triglycerides.

Saprotrophic digestion. Many fungi and bacteria secrete extracellular enzymes onto their food source (e.g. dead leaves, bread, rotting wood), digest the macromolecules externally and absorb the small soluble products. This external digestion is the basis of decomposition in ecosystems.

In all cases the enzyme is synthesised inside the cell (on ribosomes attached to the rough ER) regardless of whether it later acts inside or outside the cell.

Cambridge 9700 expects you to be able to plan and interpret an enzyme experiment. The classic intracellular-enzyme practical uses catalase:

Source of catalase. Crushed potato discs, yeast suspension or liver homogenate are all rich in catalase.

Reaction. $2\text{H}_2\text{O}_2 \rightarrow 2\text{H}_2\text{O} + \text{O}_2$.

Measurement. The rate of reaction can be measured as:

• Volume of O₂ collected in a gas syringe or over water (most accurate).
• Height of foam formed when detergent is added to trap the oxygen as bubbles.
• Time to reach a fixed volume of oxygen — gives an inverse measure of rate ($1/t$).
• Change in mass as oxygen escapes from an open flask on a balance.

Independent variables to investigate: enzyme concentration, substrate concentration, temperature, pH (with buffers), inhibitor concentration.

Controlled variables include: volume of substrate, source / mass of enzyme tissue, time of reaction, particle size of enzyme source.

These methods are revisited in detail in the next subtopic ('Factors that affect enzyme action').