3. Enzymes

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.

Compare enzyme factors through the bottleneck they change: successful enzyme–substrate collisions, the number of usable active sites, or the shape and function of those sites. Enzyme rate means the initial change in product or substrate signal per unit time, not simply the final amount.

• Temperature: Below the enzyme’s optimum, warmer conditions usually increase molecular movement and successful collisions, so the initial rate rises. Above the optimum, bonds maintaining the tertiary structure can be disrupted; the active site changes shape and the rate falls sharply through denaturation.
• pH: Each enzyme has an optimum pH. Moving away from it changes interactions and charges that maintain the active site, so productive substrate binding becomes less likely; extreme pH can denature the enzyme.
• Enzyme concentration: More enzyme supplies more active sites, so initial rate can increase while sufficient substrate remains. Once substrate becomes limiting, adding enzyme no longer gives the same increase.
• Substrate concentration: More substrate increases the chance of enzyme–substrate complex formation while active sites are available. The rate then approaches a plateau when the fixed active sites are saturated.
• Inhibitor concentration: Increasing inhibitor concentration reduces the fraction of enzyme able to catalyse productively, so the initial rate falls. Whether extra substrate can offset the effect depends on the inhibitor’s binding mechanism.

For a fair factor comparison, vary one factor while controlling the others, including temperature, pH, enzyme and substrate concentrations, volumes, mixing and measurement time. Plot the chosen progress signal against time and compare initial gradients; do not infer a rate from unequal endpoint amounts or from a plateau alone.

Avoid absolute statements such as “higher temperature” or “more enzyme” always increases rate. The direction and curve shape depend on the optimum, denaturation, substrate availability, active-site saturation and inhibitor type. Exact optimum values are enzyme-specific and are not assumed here.

On a graph of initial reaction rate, v, against substrate concentration, [S], Vmax is the capacity ceiling approached when the enzyme’s active sites are saturated. Km is the substrate concentration at which the initial rate is half of Vmax.

1. Find Vmax: Inspect the high-[S] plateau or limiting value approached by the curve; this is the estimated Vmax.
2. Find half Vmax: Mark the y-value halfway between zero and Vmax.
3. Read Km: Move horizontally from half Vmax to the curve, then vertically to the [S] axis. That x-value is Km.
4. Explain the shape: At low [S], adding substrate raises productive complex formation. At high [S], most active sites are occupied, so the curve flattens toward Vmax.

Worked read-off: if a curve approaches a clear plateau, label that plateau Vmax, mark its midpoint on the rate axis, and project from the midpoint through the curve to the substrate axis. The substrate-axis reading at that intercept is Km; do not read Km at the plateau itself.

Km is not the substrate concentration at Vmax. A lower Km can indicate higher apparent affinity only when the same model and comparable conditions apply; Vmax also depends on the amount of active enzyme. Read initial-rate data and do not invent precision beyond the graph.

Initial rate comes from the earliest linear part of a product–time or substrate–time record, before substrate depletion, product accumulation or drifting conditions begin to change the rate.

1. Prepare a sensible range of one chosen factor; equilibrate temperature or pH before mixing.
2. Start every reaction in the same way and record the signal at short, equal intervals.
3. Calculate the gradient of the earliest linear section.
4. Repeat each condition, investigate anomalies and plot mean initial rate against the independent variable.

Keep the comparison fair:

• hold enzyme and substrate source, concentrations and volumes constant unless one is the independent variable;
• use buffer to control pH and a water bath to control temperature;
• use the same timing and measurement method for every treatment;
• include a blank when the method has background colour or signal.

If product concentration rises by 12 µmol dm⁻³ during the first 30 s linear interval, the initial rate is 12 ÷ 30 = 0.40 µmol dm⁻³ s⁻¹. Apply the same interval rule to every treatment.

An endpoint amount is not an initial rate. A fixed endpoint can hide an early rate difference, while uncontrolled temperature or pH can imitate a concentration effect.

Reversible inhibitors reduce enzyme activity by limiting productive enzyme–substrate complexes. The binding site determines whether adding more substrate can recover the maximum rate.

Active-site competition lowers the chance that a substrate occupies the site at a given concentration, so increasing substrate can restore productive binding and the original maximum. Alternative-site binding changes the active site on affected enzyme molecules, so increasing substrate cannot recover the lost catalytic capacity. Read Vmax and Km from matched initial-rate saturation curves, not from one inhibitor point.

End-product inhibition is a reversible control example: as the final product accumulates, it binds at an alternative site on an earlier enzyme and slows the pathway. When product concentration falls, inhibition is relieved and the pathway can resume.

The table uses the standard simplified comparison: real data must be judged from the whole curve and matched conditions. Do not identify inhibitor type from one rate value, call alternative-site binding harmless, or introduce irreversible inhibition into this objective.

A free enzyme is added to the reaction solution. An immobilised enzyme is bound to an inert, stationary, insoluble support such as alginate, while substrate is brought to the support and product is collected separately.

• Compare equal reaction times and measure product formed per unit time, not just the final product amount.
• Keep substrate concentration, pH, temperature, total volume and measurement method the same.
• Match the starting catalytic amount as closely as possible and keep support amount/size consistent when comparing immobilised preparations.
• Repeat both treatments and compare representative means; a lower observed rate may reflect access or support effects rather than an intrinsically different enzyme mechanism.

In a supported lactase process, milk is passed over alginate beads containing lactase. Lactose is hydrolysed to glucose and galactose while the enzyme remains associated with the support, allowing the product stream to be collected and the preparation reused.

Immobilisation does not make an enzyme permanently stable, guarantee a faster rate, or justify claims that enzyme can never leave the support. Judge the measured system using matched controls and the same time basis; do not invent column dimensions or other apparatus specifications.