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3. Enzymes

Syllabus
9700–2028–2029
Section
3
Level
AS

3.1 Mode of Action

Syllabus
9700–2028–2029
Topic
3.1
Level
AS

Enzymes are globular protein catalysts with specific jobs

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.

The active site guides a specific reaction pathway

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.

Induced fit improves the catalytic arrangement

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.

Measure enzyme activity as reaction progress per unit time

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.

Use calibrated colour readings to follow enzyme progress

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.

3.2 Factors Affecting Enzyme Action

Syllabus
9700–2028–2029
Topic
3.2
Level
AS

Enzyme-rate factors change collisions, active sites or shape

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 optimum, warming usually increases molecular movement and successful collisions; above it, disruption of bonds maintaining tertiary structure changes the active site and rate falls through denaturation.
  • pH: moving from an enzyme's optimum changes charges and interactions maintaining the active site; extreme pH can denature it. Use buffer solutions in an investigation.
  • Enzyme concentration: more enzyme supplies more active sites and raises initial rate while substrate remains in excess.
  • Substrate concentration: more substrate raises complex formation until the fixed active sites are saturated and rate approaches a plateau.
  • Inhibitor concentration: more inhibitor lowers the fraction of enzyme catalysing productively; whether extra substrate offsets this depends on inhibitor type.

Vary one factor while controlling the others, including temperature, pH, enzyme and substrate concentrations, volumes, mixing and measurement time. Plot progress against time and compare initial gradients; do not infer rate from unequal endpoints or a plateau alone.

Avoid statements such as 'higher temperature' or 'more enzyme' always increases rate. The curve depends on optimum, denaturation, substrate availability, saturation and inhibitor type; exact optimum values are enzyme-specific.

Measure an initial enzyme rate with a fair test

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.

initial rate=Δproduct formedΔtor−ΔsubstrateΔt\text{initial rate}=\frac{\Delta \text{product formed}}{\Delta t}\quad\text{or}\quad-\frac{\Delta \text{substrate}}{\Delta t}

  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.

Read Vmax and Km from a substrate-saturation curve

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

v=Vmax⁡2when[S]=Kmv=\frac{V_{\max}}{2}\quad\text{when}\quad [S]=K_m

  1. Estimate Vmax from the high-substrate plateau or limiting value.
  2. Mark half Vmax on the rate axis.
  3. Move horizontally to the curve, then vertically to the substrate axis; that x-value is Km.
  4. Explain the curve: at low substrate, adding substrate increases complex formation; at high substrate, active-site saturation makes rate approach Vmax.

Read Km from the substrate-axis intercept produced by the half-Vmax construction, not from the plateau. Use initial-rate data and report only the precision supported by the graph.

Km is not the substrate concentration at Vmax. A lower Km supports higher apparent affinity only for comparable enzymes under comparable conditions; Vmax also depends on active enzyme concentration.

Compare competitive and non-competitive inhibition

Reversible inhibitors reduce enzyme activity by limiting productive enzyme-substrate complexes. Their binding site determines whether adding substrate can recover the original maximum rate.

Feature Competitive inhibitor Non-competitive inhibitor
binding site competes with substrate for the active site binds at an alternative site and changes active-site function
Vmax in the standard comparison unchanged when enough substrate outcompetes inhibitor decreased because some catalytic capacity is unavailable
apparent Km in the simple model increased unchanged
substrate-response curve shifted right but approaches the same plateau approaches a lower plateau
can more substrate recover the maximum? yes, in principle no

Active-site competition lowers substrate occupancy at a given concentration, so enough substrate can restore productive binding and the original Vmax. Alternative-site binding changes the activity of affected enzyme molecules, so extra substrate cannot recover the lost capacity. Infer type from matched initial-rate curves, not one inhibitor point.

This is the standard simplified comparison for reversible competitive and non-competitive inhibition. Judge real data from the whole curve under matched conditions; do not introduce irreversible inhibition or infer type from one rate value.

Immobilised enzymes enable separation and reuse

A free enzyme is mixed through the reaction solution. In the required immobilised system, enzyme is entrapped within insoluble alginate beads; substrate diffuses into the beads and product diffuses out while the enzyme is retained.

Feature Free enzyme Enzyme entrapped in alginate
reaction system enzyme mixes directly with substrate enzyme is retained inside beads and substrate must diffuse in
observed rate direct mixing can give rapid access diffusion through beads can limit observed rate
product separation enzyme remains mixed with product product can be collected with little or no enzyme contamination
recovery and reuse enzyme recovery is difficult beads can be retained and reused
stability and control conditions act directly on dissolved enzyme immobilisation can improve tolerance and supports continuous, controlled processing

Compare product formed per unit time using matched substrate concentration, pH, temperature, total volume and measurement method. Match starting catalytic amount as closely as possible, keep bead size and number consistent, repeat both treatments and compare representative means. A lower observed rate may reflect diffusion rather than a changed catalytic mechanism.

For lactase entrapped in alginate beads, lactose-containing solution contacts the beads. Lactose is hydrolysed to glucose and galactose, products leave the beads, and the enzyme preparation can be retained for reuse.

Entrapment does not mean the enzyme is chemically bound to alginate, permanently stable or guaranteed to react faster. Judge free and immobilised systems using matched initial-rate evidence and do not assume zero enzyme leakage.

Follow the limiting step across the topic

A rate change is evidence about the current bottleneck, not an explanation by itself. Ask whether the observation is best explained by collision frequency, substrate availability, usable active-site capacity, enzyme shape or diffusion.

  • A substrate plateau shows that enzyme capacity now limits rate; the plateau approaches Vmax.
  • Half of Vmax locates Km on the substrate-concentration axis.
  • If more substrate restores the original maximum, competition for active sites is supported.
  • If the maximum remains lower, functional enzyme capacity has been reduced.
  • If immobilised enzyme appears slower, diffusion may be limiting even though reuse and separation improve.

To make a valid conclusion, change one factor, control the others, compare initial rates and stay within the tested range. A single point cannot establish a curve shape or inhibitor type.