investigate and explain the effects of the following factors on the rate of enzyme-catalysed reactions: temperature, pH (using buffer solutions), enzyme concentration, substrate concentration, inhibitor concentration

Factors that affect enzyme action

Objective

Investigate and explain the effects of the following factors on the rate of enzyme‑catalysed reactions:

• Temperature
• pH (using buffer solutions)
• Enzyme concentration
• Substrate concentration
• Inhibitor concentration

1. Temperature

Enzyme activity increases with temperature because molecular collisions become more frequent and energetic. However, above an optimum temperature the enzyme’s tertiary structure denatures, reducing activity.

Typical relationship (qualitative):

Key points:

• Rate roughly doubles for every 10 °C rise up to the optimum (Q₁₀ effect).
• Denaturation is usually irreversible; cooling does not restore activity.

2. pH (using buffer solutions)

Enzymes have an optimum pH at which the ionisation of active‑site residues is ideal for substrate binding.

Buffers maintain a constant pH during the experiment, allowing the effect of pH alone to be examined.

Typical observations:

• Acidic or alkaline extremes lead to loss of activity due to altered charge states or denaturation.
• Different enzymes have different optima (e.g., pepsin optimum ≈ pH 2, alkaline phosphatase optimum ≈ pH 9).

3. Enzyme concentration

Increasing enzyme concentration raises the number of active sites available, so the reaction rate rises proportionally until the substrate becomes limiting.

Relationship (when substrate is in excess):

\$v = k_{\text{cat}}[E]\$

Where \$v\$ is the initial rate, \$k_{\text{cat}}\$ the turnover number, and \$[E]\$ the enzyme concentration.

4. Substrate concentration

At low substrate concentrations, the rate increases sharply because more substrate molecules encounter active sites. As the enzyme becomes saturated, the rate approaches a maximum (\$V_{\max}\$).

Michaelis–Menten equation:

\$v = \frac{V{\max}[S]}{Km + [S]}\$

Key terms:

• \$V_{\max}\$ – maximum rate when all enzyme molecules are saturated.
• \$Km\$ – substrate concentration at which \$v = \frac{1}{2}V{\max}\$ (a measure of affinity).

5. Inhibitor concentration

Inhibitors reduce enzyme activity by interacting with the enzyme or the enzyme–substrate complex. Two main reversible types are considered at A‑Level.

5.1 Competitive inhibition

The inhibitor resembles the substrate and binds to the active site, preventing substrate binding.

Effect on kinetics:

\$v = \frac{V{\max}[S]}{Km\left(1+\frac{[I]}{K_i}\right)+[S]}\$

Observations:

• \$V_{\max}\$ unchanged.
• Apparent \$K_m\$ increases (requires more substrate to reach half‑maximal rate).

5.2 Non‑competitive inhibition

The inhibitor binds to an allosteric site, not the active site, and can bind whether or not substrate is present.

Effect on kinetics:

\$v = \frac{V{\max}\left(1+\frac{[I]}{Ki}\right)^{-1}[S]}{K_m+[S]}\$

Observations:

• \$K_m\$ unchanged.
• \$V_{\max}\$ decreases (maximum rate is lowered).

Summary of factor effects

Practical considerations for A‑Level investigations

1. Use a reliable assay (e.g., spectrophotometric measurement of product formation).
2. Maintain all variables constant except the one being investigated.
3. Prepare buffer solutions covering the desired pH range (e.g., acetate, phosphate, Tris).
4. Record initial rates (linear portion of the reaction curve) to avoid complications from product inhibition.
5. Repeat each measurement at least three times to ensure reproducibility.