Factors Affecting Enzyme Activity

Enzymes are remarkably effective, but they are also fussy: change their conditions and they speed up, slow down, or stop working altogether. This lesson, part of Topic B1 of OCR Gateway Science A, examines the three factors you must know — temperature, pH and substrate concentration — and the crucial idea of denaturation. It then covers the required practical investigating the effect of pH on the enzyme amylase, and how to calculate a rate of reaction. This builds directly on the lock-and-key model from the previous lesson, because every effect here comes back to the shape of the active site.

By the end you should be able to describe and explain how temperature, pH and substrate concentration affect enzyme activity, explain denaturation, calculate a rate, and describe the amylase pH practical.

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A Quick Recap: Why Shape Matters

From the lock-and-key model, an enzyme only works when the substrate fits its active site. Anything that changes the shape of the active site changes how well the enzyme works. High temperature and the wrong pH can both change that shape permanently — this is denaturation.

Exam Tip: "Denatured" does not mean "killed" (enzymes are not alive) and it is not the same as "used up". Denatured means the enzyme's shape — especially its active site — has been permanently changed, so the substrate no longer fits.

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Factor 1: Temperature

Temperature has two opposing effects, which is why an enzyme has an optimum temperature where it works fastest.

• As temperature rises towards the optimum, the enzyme and substrate molecules gain kinetic energy and move faster, so they collide more often and with more energy. More successful collisions means a faster rate.
• Above the optimum, the heat makes the enzyme vibrate so much that bonds holding its shape break. The active site changes shape and the substrate no longer fits — the enzyme is denatured and the rate falls sharply, eventually to zero.

For many human enzymes the optimum is around 37 °C (body temperature). The shape of the temperature graph is therefore a rise to a peak, then a steep fall.

Rate of an enzyme reaction against temperature (typical data):

Temperature (°C)	Relative rate
10	2
20	4
30	7
37	10 (optimum)
45	5
55	1 (denatured)

Notice the rate rises to a peak at the optimum then drops away as the enzyme denatures — it is not symmetrical, because the fall is due to permanent denaturation, not simply fewer collisions.

Exam Tip: The rise and the fall have different causes. The rise is due to more frequent, higher-energy collisions; the fall is due to denaturation (the active site changing shape). State both causes separately for full marks.

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Factor 2: pH

Each enzyme also has an optimum pH at which it works fastest. Move the pH too far from the optimum in either direction and the enzyme denatures: the change in acidity or alkalinity alters the bonds that hold the active site's shape, so the substrate no longer fits.

Different enzymes have different optimum pH values to suit where they work in the body:

Enzyme	Where it works	Approximate optimum pH
Amylase	Mouth (saliva) and small intestine	About pH 7 (neutral)
Pepsin (a protease)	Stomach	About pH 2 (very acidic)
Lipase	Small intestine	Slightly alkaline (about pH 8)

Pepsin's low optimum suits the acidic stomach, while amylase prefers the near-neutral mouth — a neat link between an enzyme's properties and its location.

Exam Tip: Either side of the optimum pH the rate falls because the enzyme is denatured — the active site changes shape. Do not write that the enzyme is "used up" or that "the substrate denatures".

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Factor 3: Substrate Concentration

Increasing the substrate concentration generally speeds up the reaction — up to a point.

• At low substrate concentration, there are few substrate molecules, so collisions with active sites are infrequent. Adding more substrate gives more frequent collisions and a faster rate.
• At high substrate concentration, all the active sites are working as fast as they can — the enzymes are saturated. Adding still more substrate makes no further difference, so the rate levels off (plateaus).

The graph therefore rises and then flattens. (Note: this plateau is not denaturation — the enzymes are simply all occupied.)

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Calculating Rate of Reaction

OCR expects you to calculate a rate from experimental data. Rate is how much changes per unit time:

$\text{rate} = \frac{\text{change in quantity}}{\text{time taken}}$rate=time takenchange in quantity​

Worked Example 1 — Rate of product formed

In an enzyme experiment, $24 \text{ cm}^3$24 cm3 of oxygen is produced in $40 \text{ s}$40 s. Calculate the mean rate of reaction.

$\text{rate} = \frac{\text{volume of gas}}{\text{time}} = \frac{24 \text{ cm}^3}{40 \text{ s}} = 0.6 \text{ cm}^3/\text{s}$rate=timevolume of gas​=40 s24 cm3​=0.6 cm3/s

Answer: $0.6 \text{ cm}^3/\text{s}$0.6 cm3/s (cubic centimetres per second).

Common error: dividing time by volume instead of volume by time. Rate is always the quantity per unit time, so time goes on the bottom.

Worked Example 2 — Rate from a time-to-complete

A starch solution is fully broken down by amylase in $50 \text{ s}$50 s. A common way to express the rate is $\dfrac{1}{\text{time}}$time1​. Calculate this rate.

$\text{rate} = \frac{1}{\text{time}} = \frac{1}{50 \text{ s}} = 0.02 \text{ s}^{-1}$rate=time1​=50 s1​=0.02 s−1

Answer: $0.02 \text{ s}^{-1}$0.02 s−1 (per second). A larger value of $\frac{1}{\text{time}}$time1​ means a faster reaction, because the reaction finished in less time.

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Required Practical: Effect of pH on Amylase

A core practical of Topic B1 investigates how pH affects the breakdown of starch by amylase. The clever part is using iodine solution as an indicator: iodine turns blue-black in the presence of starch, but stays orange/brown once the starch has been digested. So the time taken for the blue-black colour to disappear tells you how fast the amylase worked.

flowchart TD
  A["Set up buffer solutions at a range of pH values<br/>(e.g. pH 4, 5, 6, 7, 8)"] --> B["Place drops of iodine in the wells of a spotting tile"]
  B --> C["Mix starch, amylase and one buffer in a test tube;<br/>start the timer"]
  C --> D["Every 10 s, take a drop of the mixture<br/>and add it to a fresh well of iodine"]
  D --> E["Note the time when iodine no longer turns blue-black<br/>(starch fully broken down)"]
  E --> F["Repeat for each pH; control variables<br/>(temperature, volumes, concentrations)"]

Method points the exam rewards:

• Use a water bath to keep the temperature constant (a key control variable) so you are testing pH alone.
• Control the volume and concentration of starch and amylase, and the time interval between samples.
• The end point is when the iodine stays orange/brown (no blue-black) — the starch has been digested.
• The pH at which the colour disappears fastest is the optimum pH for amylase (around neutral).
• The pH is controlled by adding a buffer solution, which keeps the pH steady at a chosen value throughout the reaction; without a buffer the pH could drift and spoil the test.

Typical results:

pH	Time for starch to disappear (s)	Rate = $1/\text{time}$1/time (s⁻¹)
4	120	0.0083
5	90	0.0111
6	60	0.0167
7	40	0.0250
8	75	0.0133