Factors Affecting Enzyme Activity
This lesson focuses in depth on the factors that affect enzyme activity and covers Core Practical 3 — investigating the effect of pH on the rate of enzyme activity — as required by the Edexcel GCSE Biology specification (1BI0), Topic 1. You need to be able to describe, explain, and analyse experimental results relating to enzyme function.
Recap: Key Factors
Three main factors affect the rate of enzyme-controlled reactions:
- Temperature
- pH
- Substrate concentration
Additionally, enzyme concentration can affect the rate, though this is less commonly tested.
Temperature and Enzyme Activity — In Detail
Below the Optimum Temperature
- At low temperatures, enzyme and substrate molecules have low kinetic energy.
- They move slowly and collide less frequently.
- Fewer enzyme-substrate complexes form per unit time.
- The rate of reaction is slow.
At the Optimum Temperature
- For most human enzymes, the optimum temperature is approximately 37°C.
- Molecules have sufficient kinetic energy to collide frequently.
- The maximum number of enzyme-substrate complexes form per unit time.
- The rate of reaction is at its highest (peak of the graph).
Above the Optimum Temperature
- Increasing temperature beyond the optimum causes the bonds that maintain the enzyme's 3D shape to vibrate excessively and break.
- The active site changes shape — this is denaturation.
- The substrate can no longer fit into the active site.
- Fewer enzyme-substrate complexes form, and the rate of reaction decreases rapidly.
- At very high temperatures, the enzyme is fully denatured and the rate of reaction is zero.
Temperature Graph — Key Features
| Feature | Description |
|---|
| Shape | Asymmetric curve (rises gradually, drops steeply) |
| Optimum | Peak of the curve (~37°C for human enzymes) |
| Below optimum | Rate increases as kinetic energy increases |
| Above optimum | Rate drops sharply due to denaturation |
| Key label | Mark the optimum temperature on the x-axis |
Exam Tip: The graph is NOT symmetrical. The drop above the optimum is much steeper than the rise below it because denaturation happens rapidly and irreversibly. If you draw this graph in an exam, make sure the right side of the peak is steeper than the left side.
pH and Enzyme Activity — In Detail
Optimum pH
- Each enzyme has a specific optimum pH at which it works most efficiently.
- At the optimum pH, the active site shape is maintained perfectly, allowing maximum enzyme-substrate complex formation.
| Enzyme | Location | Optimum pH |
|---|
| Pepsin | Stomach | ~pH 2 (acidic) |
| Salivary amylase | Mouth | ~pH 7 (neutral) |
| Pancreatic lipase | Small intestine | ~pH 8–9 (alkaline) |
| Catalase | Most cells | ~pH 7 (neutral) |
How pH Affects Enzyme Activity
- H⁺ ions (in acidic conditions) and OH⁻ ions (in alkaline conditions) interfere with the ionic and hydrogen bonds that maintain the enzyme's 3D shape.
- At extreme pH values (far from the optimum), these bonds break, the active site changes shape, and the enzyme is denatured.
- Even small changes in pH can reduce the rate of reaction significantly.
pH Graph — Key Features
- A narrow bell curve centred on the optimum pH.
- Rate drops on both sides of the optimum.
- At extreme pH values (very low or very high), the enzyme is denatured and the rate is zero.
Exam Tip: Different enzymes have different optimum pH values. If an exam question tells you an enzyme works best at pH 2, this does NOT mean all enzymes work best in acidic conditions — only that particular enzyme does.
Substrate Concentration — In Detail
Low Substrate Concentration
- Few substrate molecules are available.
- Enzyme active sites are mostly unoccupied.
- The rate of reaction is low but increases as more substrate is added.
Increasing Substrate Concentration
- More substrate molecules means more frequent collisions with enzyme active sites.
- More enzyme-substrate complexes form per unit time.
- The rate of reaction increases proportionally at first.
High Substrate Concentration (Plateau)
- Eventually, all enzyme active sites are occupied at any given moment.
- The enzymes are said to be saturated.
- Adding more substrate does not increase the rate because there are no free active sites to accept additional substrate molecules.
- The rate of reaction plateaus (levels off).
- The rate can only be increased further by adding more enzyme.
Substrate Concentration Graph — Key Features
| Feature | Description |
|---|
| Shape | Rises steeply at first, then gradually levels off to a plateau |
| Initial region | Rate is proportional to substrate concentration |
| Plateau region | All active sites are occupied — enzyme is saturated |
| How to increase rate beyond plateau | Add more enzyme molecules |
Enzyme Concentration
Although less commonly tested, enzyme concentration also affects the rate:
- Increasing enzyme concentration (while substrate is in excess) increases the rate because there are more active sites available for substrate to bind to.
- If substrate runs out, adding more enzyme will have no effect — there is not enough substrate.
Core Practical 3: Investigating the Effect of pH on Enzyme Activity
This is a key Edexcel Core Practical that you must be able to describe in detail.
Aim
To investigate how pH affects the rate of reaction of the enzyme amylase on its substrate starch.
Principle
Amylase breaks down starch into maltose (a sugar). We can track the disappearance of starch using iodine solution, which turns blue-black in the presence of starch and remains brown/yellow when starch is absent.
Equipment
- Amylase solution (enzyme)
- Starch solution (substrate)
- Buffer solutions at different pH values (e.g. pH 2, 4, 6, 7, 8, 10)
- Iodine solution
- White spotting tile (dimple tile)
- Stopwatch
- Syringes or pipettes (for measuring volumes)
- Water bath at a constant temperature (e.g. 35°C)
- Test tubes and test tube rack
- Dropping pipette
Method
- Set up a water bath at a constant temperature (e.g. 35°C) to control temperature as a variable.
- Label test tubes with the different pH values.
- In each test tube, add 2 cm³ of starch solution and 1 cm³ of the appropriate buffer solution.
- Add 1 cm³ of amylase solution to the first test tube. Start the stopwatch immediately.
- Place drops of iodine solution into the wells of a white spotting tile (one drop per well).
- Every 30 seconds, use a dropping pipette to transfer a small sample from the test tube to the next iodine drop on the spotting tile.
- Record the time taken for the iodine to stop turning blue-black — this indicates that all the starch has been broken down.
- Repeat for each pH value.
- Repeat the entire experiment at least three times and calculate a mean time for each pH.
Variables
| Variable | Details |
|---|
| Independent variable | pH (changed by using different buffer solutions) |
| Dependent variable | Time taken for starch to be completely digested (iodine no longer turns blue-black) |
| Control variables | Temperature (water bath), volume and concentration of amylase, volume and concentration of starch, volume of buffer solution |
Expected Results
- At the optimum pH (around pH 7 for amylase), starch is digested most quickly (shortest time for iodine to stop changing colour).
- At pH values above and below the optimum, the time increases (reaction is slower).
- At extreme pH values (e.g. pH 2 or pH 10), the starch may never be fully digested because the enzyme is denatured.
Calculating Rate of Reaction
Rate of reaction = 1 ÷ time
For example, if starch is fully digested in 120 seconds:
Rate = 1 ÷ 120 = 0.0083 s⁻¹
A higher rate value means a faster reaction.
Exam Tip: When describing this practical, always state: (1) the independent, dependent, and control variables, (2) the method for tracking starch breakdown (iodine on a spotting tile), (3) the need for repeats and calculating a mean, and (4) how to calculate the rate (1 ÷ time). This covers all the marks.
Analysing Enzyme Experiments
Common Graph Questions
You may be asked to: