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‑catalysed reactions

Learning objective (AS & A‑Level Biology 9700 – Topic 3 Enzymes – 3.2)

Investigate and explain how the following six factors influence the rate of an enzyme‑catalysed reaction:

• Temperature
• pH (using buffer solutions)
• Enzyme concentration
• Substrate concentration
• Reversible inhibitor concentration
• Immobilised enzymes (required by the syllabus)

Recall box – why the factors matter

All effects ultimately stem from two core ideas introduced in Topic 3.1:

• Enzyme specificity: only substrates that fit the active‑site geometry can bind.
• Active‑site geometry: maintained by weak bonds (hydrogen bonds, ionic interactions, van der Waals forces). Any factor that alters these bonds changes the shape or charge of the active site and therefore the reaction rate.

1. Temperature

• General trend: Rate rises as temperature increases because kinetic energy ↑ → more frequent and energetic collisions.
• Q₁₀ effect: Up to the optimum, the rate roughly doubles for every 10 °C rise.
• Optimum temperature: Enzyme‑specific (e.g., ≈ 37 °C for most human enzymes). At this point the tertiary structure is stable and the active‑site geometry is ideal.
• Above the optimum: Heat breaks weak bonds → denaturation → loss of active‑site shape → rapid fall in activity. Denaturation is usually irreversible; cooling does not restore activity.

2. pH (using buffer solutions)

• Purpose of buffers: Keep pH constant throughout the assay so that any change in rate is due solely to the set pH, not to pH drift caused by the reaction.
• Optimum pH: The ionisation state of key active‑site residues (e.g., –COOH, –NH₂, –SH) is ideal for substrate binding at a particular pH.
• Effect of extremes: Excess H⁺ or OH⁻ alters charge states or disrupts tertiary structure → activity falls.
• Typical examples:

  • Pepsin – optimum ≈ pH 2 (stomach)
  • Human salivary amylase – optimum ≈ pH 7 (mouth)
  • Alkaline phosphatase – optimum ≈ pH 9 (intestine)

3. Enzyme concentration

When substrate is in excess, the initial rate (v) is directly proportional to the amount of active enzyme present.

• Increasing [E] adds more active sites → higher turnover.
• Linear relationship persists until substrate becomes limiting; then the curve plateaus.

4. Substrate concentration

The relationship is hyperbolic and is described by the Michaelis–Menten model.

Key terms

• V_max – maximum rate when every enzyme molecule is saturated with substrate.
• K_m – substrate concentration at which v = ½ V_max; a measure of enzyme–substrate affinity (low K_m = high affinity).

5. Reversible inhibitor concentration

5.1 Types of reversible inhibition

• Competitive: Inhibitor (I) resembles the substrate and binds to the active site, preventing substrate binding.
• Non‑competitive: Inhibitor binds to an allosteric site; it can bind whether or not substrate is present, reducing the number of functional enzyme molecules.

5.2 Kinetic effects

Competitive inhibition

• V_max unchanged – at very high [S] the substrate out‑competes the inhibitor.
• Apparent K_m increases (more substrate needed to reach half‑maximal rate).

Non‑competitive inhibition

• K_m unchanged – affinity for substrate is not altered.
• V_max decreases – a fraction of enzyme molecules is permanently inactivated by the inhibitor.

5.3 Brief note on irreversible inhibition (AO3 relevance)

Irreversible inhibitors form covalent bonds with the enzyme (e.g., aspirin with cyclo‑oxygenase). They permanently reduce the amount of active enzyme, lowering V_max without affecting K_m. Although not examined for AO2, this knowledge is useful for evaluation questions.

6. Immobilised enzymes

• Definition: Enzymes are fixed to a solid support (e.g., calcium‑alginate beads, silica gel, polymer membranes) while retaining catalytic activity.
• Why use them? Easy separation from reaction mixture, re‑use of the catalyst, and often greater stability under extreme pH or temperature.
• Effect on activity: Initial rates are usually lower than for the free enzyme because substrate must diffuse into the matrix; however, the total catalytic output over many cycles can be higher.
• Typical investigation (catalase example):

  1. Prepare calcium‑alginate beads containing catalase (mix enzyme with 2 % Na‑alginate, drop into 0.1 M CaCl₂).
  2. Add a fixed volume of H₂O₂ (substrate) to each tube containing a known mass of beads.
  3. Collect the volume of O₂ released in a gas‑collecting syringe over a fixed time (e.g., 30 s).
  4. Vary the bead mass (enzyme concentration) while keeping H₂O₂ concentration constant; plot initial rate vs. bead mass.

7. Practical data analysis (AO2 & AO3)

7.1 Linearising Michaelis–Menten data

• Lineweaver–Burk plot: 1/v against 1/[S] → y‑intercept = 1/V_max, x‑intercept = –1/K_m.
• Eadie–Hofstee plot: v against v/[S] → slope = –K_m, y‑intercept = V_max.
• Limitations: Over‑weighting of low‑[S] points, propagation of experimental error, and the assumption that steady‑state conditions are met.

7.2 Common sources of error (AO3)

• Inaccurate temperature control (water‑bath fluctuations).
• Poor buffer capacity → pH drift during the assay.
• Enzyme denaturation on repeated use or prolonged assay time.
• Pipetting errors when preparing dilution series of substrate, enzyme or inhibitor.
• Instrumental lag (e.g., spectrophotometer response time) affecting the measurement of initial rates.

7.3 Evaluation tips

• Always record the linear (initial) portion of the reaction curve.
• Repeat each measurement ≥ 3 times; present mean ± standard deviation.
• Compare observed optima (temperature, pH) with literature values and discuss possible reasons for any discrepancy.
• For inhibition studies, demonstrate reversibility by removing the inhibitor (e.g., dialysis) and showing activity recovery.
• When using immobilised enzymes, comment on diffusion limitations and the trade‑off between initial rate and re‑usability.

8. Summary table – effect of each factor on reaction rate

9. Practical checklist for A‑Level investigations

1. Choose a reliable assay (e.g., spectrophotometric measurement of product formation, gas‑evolution for catalase, colour change for p‑nitrophenyl‑phosphate).
2. Keep all variables constant except the one being investigated.
3. Prepare a series of buffer solutions covering the required pH range (acetate, phosphate, Tris) and verify their pH with a calibrated pH meter.
4. Record initial rates (the linear portion of the reaction curve) to avoid complications from product inhibition.
5. Perform each measurement at least three times; calculate mean ± SD and include a brief statistical comment.
6. For kinetic parameters, plot the data (Lineweaver–Burk or Eadie–Hofstee) and extract V_max and K_m with appropriate units.
7. Identify sources of error, discuss their likely impact on the results, and suggest realistic improvements (AO3).
8. When studying immobilised enzymes, note the mass of support used, any pre‑washing steps, and the number of reuse cycles tested.