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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.