Investigate and describe the effect of changes in temperature and pH on enzyme activity.

5.1 Enzymes

Learning objective

Investigate and describe the effect of changes in temperature and pH on enzyme activity.

Core description of enzymes (syllabus requirement)

• Enzymes are biological catalysts; they increase the rate of a reaction without being consumed.
• Enzymes are proteins. Their three‑dimensional shape determines the position of the active site where the substrate binds.
• Enzymes lower the activation energy of a reaction, allowing it to proceed faster at a given temperature.
• Because they are proteins, enzymes can be denatured (their shape is altered) by extreme temperature or pH, which reduces or abolishes activity.

Key factors that influence enzyme activity

• Temperature – influences kinetic energy and collision frequency. Each enzyme has an optimum temperature.
• pH – influences ionisation of amino‑acid side‑chains that maintain the enzyme’s shape and active‑site geometry. Each enzyme has an optimum pH.
• Both factors produce a characteristic bell‑shaped curve: activity rises to the optimum and then falls sharply as denaturation occurs.

Effect of temperature on enzyme activity

• Rising temperature → higher kinetic energy → more frequent enzyme‑substrate collisions → reaction rate increases.
• Maximum activity occurs at the enzyme’s optimum temperature (≈ 37 °C for most human enzymes).
• Above the optimum, hydrogen bonds and other weak interactions that maintain secondary and tertiary structure break, the enzyme denatures, and activity falls rapidly.
• Very low temperatures give few collisions, so the reaction rate is also low.
• Denaturation at high temperature is usually irreversible; the enzyme cannot regain its original shape when cooled.

Effect of pH on enzyme activity

• pH changes the ionisation of amino‑acid residues that hold the enzyme’s shape and line the active site.
• Each enzyme works best at a specific pH (e.g., pepsin ≈ pH 2, amylase ≈ pH 7).
• Moving away from the optimum alters the charge of side‑chains, distorts the active site and reduces activity.
• Very acidic or very alkaline conditions cause permanent denaturation of the protein.

Practical investigation – effect of temperature

Apparatus, materials and reagents

Safety note

• Handle hot water baths with tongs or heat‑proof gloves.
• Hydrogen peroxide can irritate skin and eyes – wear goggles and avoid splashes.
• Dispose of enzyme extracts according to school safety guidelines.

Variables

• Independent variable: Temperature of the reaction mixture.
• Dependent variable: Volume of O₂ produced in 1 min (rate of reaction, mL O₂ min⁻¹).
• Controlled variables: Enzyme source, substrate concentration, volume of substrate, volume of enzyme added, reaction time, and size of test tube.

Procedure (temperature)

1. Label five test tubes “0 °C”, “20 °C”, “37 °C”, “50 °C” and “70 °C”.
2. Add 5 ml of 3 % hydrogen peroxide to each tube.
3. Place the tubes in the corresponding water bath for 2 min to equilibrate.
4. Add 0.5 ml of the catalase extract, start the stopwatch and immediately begin collecting the released oxygen (e.g., by water displacement).
5. Record the volume of gas produced after 1 min.
6. Repeat the whole experiment twice and calculate the average volume for each temperature.

Sample temperature data

Practical investigation – effect of pH

Apparatus, materials and reagents (additional)

Variables

• Independent variable: pH of the reaction mixture.
• Dependent variable: Volume of O₂ produced in 1 min (mL O₂ min⁻¹).
• Controlled variables: Enzyme source, substrate concentration, temperature (kept at optimum, e.g., 37 °C), reaction time, volumes of substrate and enzyme.

Procedure (pH)

1. Label six test tubes “pH 2”, “pH 4”, “pH 6”, “pH 7”, “pH 8”, “pH 10”.
2. Add 5 ml of the appropriate buffer to each tube.
3. Add 5 ml of 3 % hydrogen peroxide (final volume ≈ 10 ml).
4. Place all tubes in a water bath set at the enzyme’s optimum temperature (≈ 37 °C) for 2 min.
5. Add 0.5 ml of the catalase extract, start the stopwatch and collect O₂ for 1 min.
6. Record the volume of gas released, repeat twice and calculate the average for each pH.

Sample pH data (catalase)

Graphical representation (suggested)

Analysis of results (exam‑style points)

• Explain why activity rises with temperature up to the optimum (higher kinetic energy → more collisions, lower activation energy).
• Describe the structural changes that occur when temperature exceeds the optimum (breakdown of hydrogen bonds, loss of tertiary structure → irreversible denaturation).
• Link pH changes to ionisation of side‑chains in the active site and the resulting distortion of the enzyme’s shape.
• State that denaturation is generally irreversible because the protein cannot refold correctly without assistance.
• Relate the observations to the concepts of activation energy and collision theory.

Key vocabulary

• Enzyme
• Protein
• Active site
• Substrate
• Optimum temperature
• Optimum pH
• Denaturation
• Catalysis
• Reaction rate
• Activation energy
• Collision theory

Practice exam questions

1. Explain why the rate of reaction catalysed by amylase increases when the temperature is raised from 20 °C to 37 °C.
2. Describe the molecular changes that occur to an enzyme when it is heated to 70 °C.
3. Given a graph of enzyme activity against pH, identify the optimum pH and explain why activity declines at more extreme values.
4. Design a simple experiment to test the effect of pH on the activity of the enzyme pepsin. Include apparatus, variables, safety considerations and how you would record the results.
5. The temperature investigation uses a water‑bath at 70 °C. Suggest two improvements to make the method more reliable and explain why they would improve the results.

Real‑world application

• Enzymes are added to laundry detergents because they work best at moderate temperatures (≈ 30‑40 °C) and near‑neutral pH. This allows stains to be removed efficiently while reducing the energy required to heat water, giving economic and environmental benefits.

Summary

Enzymes are protein catalysts that function most efficiently at a specific temperature and pH. Within these optimum ranges, increasing temperature raises kinetic energy and collision frequency, while the correct pH maintains the ionisation state of amino‑acid residues in the active site. Outside the optimum, low temperature reduces collision frequency, and extreme temperature or pH values cause irreversible denaturation of the protein, leading to a sharp fall in catalytic activity. Understanding these principles is essential for interpreting experimental data and for applications such as food processing, medicine, biotechnology and household products.