Enzyme Activity – Factors and Reversible Inhibition
Learning Objectives (Cambridge AS & A‑Level Biology 9700)
- AO1: Describe the factors that affect enzyme activity and explain how reversible (competitive and non‑competitive) inhibitors influence the rate of reaction.
- AO2: Analyse experimental data (e.g. Michaelis–Menten and Lineweaver‑Burk plots) to determine the kinetic effect of different inhibitors and other factors.
- AO3: Plan, carry out and evaluate a practical investigation that investigates the effect of inhibitors (and, where appropriate, temperature, pH or concentrations) on enzyme activity.
1. Factors that Influence Enzyme Activity (AO1)
Enzymes are biological catalysts; their activity can be altered by a range of internal and external factors. The syllabus expects you to be able to name and explain the effect of each factor.
| Factor | Typical Effect on Reaction Rate | Reason |
|---|
| Temperature | Rate ↑ up to an optimum, then ↓ sharply | Higher temperature increases kinetic energy → more collisions; above optimum denaturation destroys active‑site geometry. |
| pH | Rate ↑ to an optimum, then ↓ | pH alters ionisation of active‑site residues; extreme pH causes denaturation. |
| Enzyme concentration | Rate ↑ (linear) when substrate is in excess | More enzyme molecules mean more active sites available. |
| Substrate concentration | Rate ↑ rapidly then plateaus (Vmax) | At high [S] all enzyme molecules are saturated (ES complex formation reaches maximum). |
| Reversible inhibitors (competitive, non‑competitive, uncompetitive) | Change apparent Km and/or Vmax without destroying the enzyme | Inhibitor binds non‑covalently; effect depends on binding site and whether it can bind to ES. |
| Irreversible inhibitors | Permanent loss of activity (Vmax ↓, Km unchanged) | Covalent modification of active‑site residues prevents substrate binding. |
| Allosteric regulation (activators or inhibitors) | Can increase or decrease Vmax; often changes Km | Binding at a site distinct from the active site induces conformational change. |
| Cofactors / co‑enzymes | May be required for activity; absence → ↓ rate | Provide essential groups (e.g., metal ions, vitamins) for catalysis. |
2. Reversible Inhibitors – Competitive and Non‑competitive (AO1)
2.1 General Features
- Bind to the enzyme by non‑covalent forces (hydrogen bonds, ionic interactions, Van‑der‑Waals).
- The binding is reversible – the inhibitor can dissociate, restoring activity.
- They alter the kinetic parameters Km (affinity) and/or Vmax (maximum rate) in characteristic ways.
2.2 Competitive Inhibition
- The inhibitor has a structure similar to the substrate and competes for the active site.
- It can bind only to the free enzyme (E); it cannot bind to the enzyme–substrate complex (ES).
- Increasing substrate concentration can out‑compete the inhibitor, restoring the original rate.
- Kinetic effect: Vmax is unchanged; apparent Km increases.
Modified Michaelis–Menten equation
\$v=\frac{V{\max}[S]}{Km\left(1+\frac{[I]}{K_i}\right)+[S]}\$
where [I] = inhibitor concentration and Ki = inhibition constant.
2.3 Non‑competitive Inhibition
- The inhibitor binds to an allosteric site that is distinct from the active site.
- It can bind to both free enzyme (E) and the ES complex, forming EI and ESI.
- Increasing substrate concentration cannot overcome the inhibition because the active site remains accessible but the enzyme’s catalytic efficiency is reduced.
- Kinetic effect: Vmax decreases; apparent Km is unchanged.
Modified Michaelis–Menten equation
\$v=\frac{V{\max}\left(1+\frac{[I]}{Ki}\right)^{-1}[S]}{K_m+[S]}\$
2.4 Comparison at a Glance
| Feature | Competitive Inhibition | Non‑competitive Inhibition |
|---|
| Binding site | Active site (substrate‑like) | Allosteric site (different) |
| Enzyme form bound | Only free enzyme (E) | Both free enzyme (E) and ES complex |
| Effect on Vmax | No change | Decreases |
| Effect on apparent Km | Increases | No change |
| Can be overcome by more substrate? | Yes | No |
| Typical example | Methotrexate vs dihydrofolate reductase (substrate analogue) | Lead (Pb²⁺) inhibiting δ‑aminolevulinic acid dehydratase (allosteric binding) |
2.5 Real‑World Relevance (Biological, Social & Economic Context)
- Drug design: Many pharmaceuticals are competitive inhibitors (e.g., statins inhibit HMG‑CoA reductase) or non‑competitive inhibitors (e.g., allosteric modulators of neurotransmitter receptors).
- Pesticide resistance: Mutations that reduce inhibitor binding (e.g., altered acetylcholinesterase) illustrate the importance of understanding kinetic inhibition.
- Toxicology: Heavy‑metal poisoning often involves non‑competitive inhibition of key metabolic enzymes.
3. Assumptions of the Michaelis–Menten Model (AO2)
- Steady‑state: The concentration of the ES complex remains constant during the initial phase of the reaction.
- Substrate excess: [S] ≫ [E]; substrate concentration does not change appreciably during the short measurement period.
- Single substrate, single active site: The model applies to simple one‑substrate reactions.
- Negligible reverse reaction and product inhibition: Measurements are taken before significant product accumulates.
4. Practical Investigation – Effects of Inhibitors (and optionally temperature/pH) (AO2 & AO3)
4.1 Planning Template
| Section | Content to Include |
|---|
| Aim | To determine how a competitive and a non‑competitive inhibitor affect the kinetic parameters (Km and Vmax) of catalase‑catalysed decomposition of hydrogen peroxide. |
| Hypothesis | Azide (competitive) will increase the apparent Km but not change Vmax; lead ions (non‑competitive) will lower Vmax with no change in Km. |
| Variables | - Independent: Inhibitor type and concentration; substrate concentration ([H₂O₂]).
- Dependent: Initial rate of O₂ evolution (ΔV/Δt) or decrease in absorbance at 240 nm.
- Controlled: Temperature (e.g., 25 °C water bath), pH (phosphate buffer, pH 7.0), enzyme amount (fixed mass of catalase), volume of reaction mixture.
|
| Materials | Catalase solution (e.g., from potato), H₂O₂ (various concentrations), sodium azide solution, Pb(NO₃)₂ solution, phosphate buffer (pH 7.0), colourimetric reagent (e.g., KI/TiOSO₄), spectrophotometer or gas‑collection apparatus, water bath, pipettes, test tubes. |
| Method (outline) | - Prepare a series of H₂O₂ solutions (0.5, 1.0, 2.0, 4.0 mM) in buffer.
- For each substrate concentration set up three reactions: control, + azide (1 mM), + Pb²⁺ (0.5 mM).
- Add a fixed volume of catalase to start the reaction; immediately record the change in absorbance at 240 nm every 10 s for 1 min (or collect O₂ gas for 30 s).
- Repeat each assay at least three times to ensure reliability.
|
| Safety | Handle H₂O₂ (oxidiser) with gloves; azide is toxic – work in a fume hood; lead salts are hazardous – wear lab coat and dispose of waste according to regulations. |
| Repeatability | Perform each measurement in triplicate; randomise the order of substrate concentrations to minimise systematic error. |
4.2 Data Analysis Checklist (AO2)
- Convert absorbance change to concentration change using Beer‑Lambert law (or calculate gas volume to moles of O₂).
- Calculate the initial rate (v) for each [S] (slope of the first linear segment).
- Plot a Michaelis–Menten curve (v vs [S]) for each condition.
- Construct a Lineweaver‑Burk plot (1/v vs 1/[S]) and determine:
- y‑intercept = 1/Vmax
- x‑intercept = –1/Km
- Compare the slopes and intercepts:
- Competitive: lines intersect on the y‑axis (same 1/Vmax, larger slope → higher apparent Km).
- Non‑competitive: lines intersect on the x‑axis (same –1/Km, higher y‑intercept → lower Vmax).
- Calculate % error between observed and literature values of Km and Vmax.
4.3 Evaluation Prompts (AO3)
- How might oxygen bubbles affect the spectrophotometric measurement? Suggest a method to minimise this (e.g., use a gas‑tight cuvette).
- Discuss the reliability of the colourimetric assay at high H₂O₂ concentrations (possible substrate inhibition).
- Identify sources of random error (pipetting, temperature fluctuations) and systematic error (incorrect inhibitor concentration).
- Propose an improvement: e.g., use a stopped‑flow apparatus for more precise initial‑rate determination.
- Explain how the experiment could be extended to investigate temperature or pH effects while keeping inhibitor concentration constant.
5. Summary (AO1)
- Enzyme activity is modulated by temperature, pH, enzyme/substrate concentrations, cofactors, and inhibitors.
- Reversible inhibitors bind non‑covalently:
- Competitive: bind active site → increase apparent Km, Vmax unchanged; effect can be overcome by excess substrate.
- Non‑competitive: bind allosteric site → decrease Vmax, Km unchanged; substrate cannot overcome inhibition.
- Understanding these kinetic signatures enables prediction of drug action, toxicity, and metabolic regulation.
Key Points to Remember (AO1)
- An increase in Km reflects reduced affinity of enzyme for substrate.
- A decrease in Vmax indicates that fewer active enzyme molecules are available for catalysis.
- Only competitive inhibition can be mitigated by raising substrate concentration; non‑competitive inhibition cannot.
- When analysing data, the pattern of line intersections on a Lineweaver‑Burk plot tells you instantly which type of reversible inhibition is present.