Enzymes don't work in isolation. Temperature, pH, and substrate concentration all push enzyme activity up or down — sometimes with dramatic consequences. Understanding these factors is essential for explaining how cells control their chemistry.
A student sets up three test tubes containing starch solution and amylase enzyme:
After 5 minutes, she adds iodine solution to each tube. Iodine turns blue-black in the presence of starch.
Predict the colour of each tube and explain your reasoning:
Come back to this at the end of the lesson.
Core Content
Every enzyme operates best within a specific range of conditions. Push too far outside this range, and the enzyme stops working — sometimes permanently. The three key environmental factors are:
Affects kinetic energy of molecules and enzyme shape
Key concept: Denaturation at high temps
Affects ionisation of amino acid side chains
Key concept: Each enzyme has optimal pH
Determines collision frequency with active sites
Key concept: Saturation at high [S]
As temperature increases, molecules move faster. This means more frequent collisions between enzyme and substrate, so reaction rate increases — up to a point.
At the optimum temperature, the enzyme works at maximum efficiency. For most human enzymes, this is around 37°C (body temperature).
Beyond the optimum, the kinetic energy becomes too great. The bonds holding the enzyme's tertiary structure break, and the active site changes shape. This is denaturation — and it's usually permanent.
At very low temperatures, enzymes work slowly but are not denatured. The enzyme retains its shape — there's just less kinetic energy for collisions. This is why freezing preserves food: enzymes still exist, but they work too slowly to cause spoilage.
pH affects the ionisation state of amino acid side chains, especially those in the active site. Changing pH can alter the charges that hold the active site in its specific conformation — or change the charges involved in binding substrate.
Different enzymes have different pH optima based on where they work:
Like temperature, extreme pH causes denaturation. The enzyme's tertiary structure unravels as ionic bonds between charged amino acids are disrupted.
At low substrate concentration, reaction rate increases linearly with [S] — more substrate means more collisions with available enzyme active sites.
However, once all active sites are occupied, adding more substrate cannot increase the rate further. The enzyme is working at Vmax (maximum velocity), and the reaction is limited by enzyme concentration, not substrate availability.
The HSC requires you to understand how to design valid experiments investigating enzyme activity. Here's a template for investigating any of the three factors:
Aim: To investigate the effect of [independent variable] on the activity of [enzyme name]
Method principles:
Common methods for measuring enzyme activity:
| Enzyme | Substrate | Measurement Method |
|---|---|---|
| Amylase | Starch | Iodine test (blue-black → yellow/brown as starch is digested) |
| Catalase | Hydrogen peroxide | Oxygen gas production (count bubbles or measure volume) |
| Protease | Protein (gelatin) | Clearing of cloudy suspension or digestion of photographic film |
Activities
A student investigated the effect of temperature on amylase activity. Her results are shown below:
Write your responses here:
Catalase is an enzyme found in liver that breaks down hydrogen peroxide (H₂O₂) into water and oxygen gas. Design an experiment to investigate how pH affects catalase activity.
In your answer, include:
Write your experimental design here:
Assessment
1. Which of the following best explains why enzyme activity decreases at temperatures above the optimum?
2. An enzyme has an optimum pH of 2. Where in the human body is this enzyme most likely to be found?
3. In an enzyme-catalysed reaction, what happens when substrate concentration increases beyond the point where all active sites are occupied?
4. Why can enzymes that have been kept at 0°C resume normal activity when warmed to 37°C, while enzymes kept at 80°C cannot?
5. Which of the following statements about enzyme denaturation is correct?
1. Explain the effect of increasing temperature on enzyme activity from 0°C to 60°C. In your answer, refer to kinetic energy, collision theory, and denaturation. (3 marks)
1 mark for explaining increased activity below optimum; 1 mark for kinetic energy/collision theory; 1 mark for explaining denaturation above optimum
2. Compare and contrast the effect of substrate concentration versus temperature on enzyme-catalysed reactions. (3 marks)
1 mark for similarity (both affect rate); 1 mark for difference (substrate doesn't change enzyme, temperature does); 1 mark for explaining saturation vs. denaturation
3. A pharmaceutical company is developing a drug that must be stored for long periods. The active ingredient is a protein-based enzyme. Explain why the company might choose to store the drug at 4°C rather than at room temperature (25°C), and why storage at -20°C might be even better for long-term stability. (3 marks)
1 mark for explaining reduced activity at 4°C; 1 mark for explaining risk of denaturation at 25°C; 1 mark for explaining minimal molecular motion at -20°C
Answers
SA1 (3 marks): As temperature increases from 0°C to approximately 37°C (the optimum), enzyme activity increases because the kinetic energy of both enzyme and substrate molecules increases, leading to more frequent collisions and more successful substrate binding (collision theory). However, beyond the optimum temperature, the increased thermal energy disrupts the weak bonds (hydrogen bonds, ionic bonds) that maintain the enzyme's tertiary structure. This causes denaturation — the active site loses its specific shape, substrate can no longer bind, and enzyme activity decreases sharply.
SA2 (3 marks): Both increasing substrate concentration and increasing temperature (up to the optimum) increase the rate of enzyme-catalysed reactions. However, substrate concentration affects only the frequency of enzyme-substrate collisions and does not change the enzyme itself — once saturation is reached (Vmax), the rate plateaus. In contrast, temperature affects the enzyme's structure directly; above the optimum, the enzyme denatures and activity decreases permanently. Additionally, while increasing substrate concentration cannot damage the enzyme, excessive temperature causes irreversible loss of function.
SA3 (3 marks): At 4°C, the enzyme maintains its tertiary structure and would function normally if warmed, but its activity is significantly reduced due to lower kinetic energy — making it suitable for short-term storage. At 25°C (room temperature), the enzyme remains active and could gradually degrade over time due to ongoing catalytic activity and potential denaturation. Storage at -20°C is superior for long-term stability because the frozen state essentially stops all molecular motion, preventing both catalytic degradation and any gradual denaturation processes that might occur even at 4°C.
Earlier you predicted the results of an amylase experiment at three temperatures. Compare your predictions with the correct answers below:
| Tube | Temperature | Expected Result | Explanation |
|---|---|---|---|
| A | 5°C | Blue-black (starch present) | Low kinetic energy — enzyme works very slowly. After only 5 minutes, most starch remains undigested. |
| B | 37°C | Yellow/brown (little/no starch) | Optimum temperature for human amylase. Rapid digestion of starch occurs. |
| C | 80°C | Blue-black (starch present) | Enzyme is denatured. The active site has lost its shape and cannot bind starch. |
Were your predictions correct? Did you recognise that both 5°C and 80°C would show starch present, but for very different reasons?