3.1 Mode of Action

An enzyme is a globular protein that acts as a biological catalyst. It speeds up a chemical reaction by providing a pathway with lower activation energy, without being used up or permanently changed.

• Substrate binding: A substrate binds temporarily at the enzyme’s specific active site, forming an enzyme–substrate complex. The folded protein gives the active site its shape and chemical environment.
• Catalysis: Interactions at the active site help the reaction reach its activated state more readily, so products form faster.
• After the reaction: Products leave and the enzyme remains available to catalyse another reaction. Lowering activation energy changes the reaction rate; it does not change the overall energy difference or the position of equilibrium.
• Where the job occurs: Intracellular enzymes are made and act inside cells. Extracellular enzymes are secreted and act outside cells, for example during digestion of large food molecules.

Catalase is an intracellular example that breaks down hydrogen peroxide, a harmful by-product of cellular reactions. Amylase is an extracellular digestive example that breaks starch into smaller sugars. The examples illustrate location and role; they do not mean an enzyme is consumed by the reaction.

“Intracellular” and “extracellular” describe where an enzyme acts, not whether an organism is single-celled or multicellular. Enzymes are proteins, so conditions that disrupt protein structure can impair activity, but the catalytic reaction itself does not use the enzyme up.

An enzyme’s active site is a three-dimensional region whose shape and chemical groups are complementary to a particular substrate. Specific binding creates a temporary enzyme–substrate complex and positions the reactants for catalysis.

1. Collision: The substrate collides with the active site at a suitable orientation and speed.
2. Binding: Complementary shape and chemical interactions hold the substrate in the active site, forming a temporary enzyme–substrate complex.
3. Catalysis: The active site helps destabilise relevant substrate bonds and provides an alternative pathway with lower activation energy, so products form more readily.
4. Release: Products leave the active site and the unchanged enzyme is ready to bind another substrate.

The protein’s tertiary structure determines the active-site shape, so a change in amino-acid sequence can change the site and enzyme specificity. Complementary binding is therefore the gate for the reaction; a collision with the wrong shape need not produce a complex or products.

The active site does not permanently lock the substrate in place, and the enzyme is not used up. Lower activation energy increases reaction rate through an alternative route; it does not change the overall energy difference or move the equilibrium. The small binding adjustment described by induced fit is developed in the next card.

The lock-and-key model is a useful first explanation of enzyme specificity because it shows a complementary substrate and active site. The induced-fit hypothesis extends this model: binding causes a small adjustment in the enzyme’s active site, and sometimes in the substrate, so the final complex is better arranged for catalysis.

• Initial recognition: The substrate is chemically and structurally compatible enough to interact with the active site; this preserves specificity.
• Small adjustment: As binding proceeds, the enzyme and active site can change shape slightly rather than behaving as perfectly rigid structures.
• Improved fit: The adjustment brings reactive groups into a more effective arrangement and helps the enzyme stabilise the reaction pathway.
• Catalytic consequence: A better enzyme–substrate arrangement increases the ability of the enzyme to catalyse the reaction; the adjustment is part of binding, not a permanent change to the whole protein.

Lock-and-key emphasises a pre-existing rigid complementarity. Induced fit keeps the idea of selective binding but explains why the active site can adjust around a suitable substrate to improve catalysis.

Induced fit is not permanent deformation or denaturation, and it does not make every molecule a suitable substrate. Card 4542 covers the general collision–complex–product process; this card explains the binding model. Rate measurement is handled separately in card 4544.

Enzyme activity is the rate of an enzyme-catalysed reaction: how much substrate is used or product is formed per unit time. In general, rate = change in measured amount ÷ time taken, using a consistent signal and time interval.

1. Choose a progress signal: Follow product formation, such as oxygen produced by catalase, or substrate disappearance, such as starch being hydrolysed by amylase.
2. Measure at defined times: Start the reaction consistently and record the signal at set intervals. For amylase, samples can be tested with iodine; starch gives a blue-black result while it remains.
3. Calculate rate: Plot progress against time when appropriate and use the gradient of the early, approximately linear section as the initial rate. A final amount or a plateau is not itself a rate.
4. Compare fairly: Keep temperature, pH, enzyme concentration, substrate concentration, volumes, mixing and timing controlled; repeat measurements and calculate a representative mean when the design requires it.

A faster reaction changes the chosen product or substrate signal more quickly, so it has a steeper initial gradient. The initial section is preferred because later progress can slow as substrate is depleted, products accumulate or another experimental limit is reached.

Do not call a single endpoint amount an enzyme rate, and do not infer a rate from unequal time intervals without accounting for time. This card defines the general progress-over-time method; the separate colorimetry card explains how a colour signal is calibrated into quantitative readings.

Colorimetry measures how much light a coloured sample absorbs or transmits. When the colour is linked to a substrate or product, the reading acts as a concentration proxy, allowing enzyme progress to be followed more objectively than judging colour by eye.

1. Set the reference: Use an appropriate blank to zero the colorimeter before measuring samples. Keep the cuvette clean and consistently oriented.
2. Calibrate the relationship: Prepare known concentrations, for example by serial dilution of a stock solution, and measure their absorbance or transmission. Plot concentration against reading to make a calibration graph.
3. Read unknowns: Measure the unknown reaction samples under the same conditions. Use the calibration relationship to estimate the corresponding substrate or product concentration.
4. Follow the rate: Take readings at defined times and plot the calibrated concentration, or a justified signal proxy, against time. The early linear gradient gives an initial rate; faster progress gives a steeper gradient.

A changing colour changes the amount of light absorbed or transmitted; calibration connects that reading to concentration; repeated timed readings then connect concentration change to enzyme rate. In a starch–amylase example, iodine colour decreases as starch is hydrolysed, so the calibrated decline in starch can be followed over time.

Absorbance or transmission is not automatically a concentration or a rate: the blank, standards, timing, cuvette handling and usable calibration range support that inference. This card does not assume a particular wavelength, unit or instrument specification.