Three factors change how fast an enzyme works: temperature, pH and substrate concentration. You need to be able to sketch all three graphs from memory and explain every part of the shape — not just the peak.
📚 What you need to know
Temperature gives a curve that rises to an optimum and then falls away sharply.
The rise is about collisions; the fall is about denaturation. Two different explanations on one graph.
pH gives a narrow peak at the optimum. Different enzymes have very different optima.
Substrate concentration gives a curve that rises then levels off when all active sites are saturated.
Rate can be followed by measuring product appearing or substrate disappearing.
In any experiment, name the independent variable, the dependent variable and the controls — and say how you measured the dependent variable.
Sketch graphs are models: you should be able to redraw a curve for changed conditions, not just copy one.
Temperature
This is the graph students draw quickly and explain badly. It has two halves, and they need completely different answers.
Draw the peak slightly left of centre and let the right-hand side drop almost vertically — a symmetrical hill is a common way to lose the shape mark.
Explaining the rising side
Higher temperature gives molecules more kinetic energy, so they move faster.
Faster movement means enzyme and substrate collide more often.
They also collide with more energy, so more of those collisions are successful.
More enzyme–substrate complexes form each second, so the rate goes up.
Explaining the falling side
Above the optimum, vibration in the molecule breaks the weak bonds holding its 3D shape.
The active site changes shape, so it is no longer complementary to the substrate.
The substrate cannot bind, no complex forms, and the rate drops.
Since denaturation is permanent, cooling the mixture down again does not bring the rate back.
If a question says “explain the shape of the graph”, it almost always wants both halves. Write about collisions for the rise and denaturation for the fall — and never mix the two round.
pH
pH measures the concentration of hydrogen ions. It runs on a logarithmic scale, so each step of one pH unit is a tenfold change in hydrogen ion concentration — pH 4 is ten times more acidic than pH 5, not slightly more.
Those hydrogen ions interfere with the charges on the R-groups that hold the enzyme’s shape together. A little way from the optimum and the shape is distorted; further away and the enzyme is denatured for good.
The curves are narrow on purpose. Move one or two pH units away from the optimum and most of the activity has already gone.
Do not assume pH 7. Pepsin works best around pH 2 because the stomach is acidic, and some bacterial enzymes work best at pH 9–10. The optimum always matches the enzyme’s normal surroundings.
Substrate concentration
Add more substrate and at first the rate climbs steeply — there are plenty of empty active sites, so extra substrate molecules find one quickly. But active sites are occupied while a reaction is happening, and there is a fixed number of them.
Eventually every active site is busy the whole time. The enzymes are working flat out and the rate cannot rise any further. That point is called saturation.
The curve flattens but never turns downwards. Extra substrate does no harm — it simply queues.
A favourite follow-up question: “how could you increase the rate once the plateau is reached?” The answer is more enzyme, because that adds more active sites. More substrate does nothing at all.
Designing the experiment
Whichever factor you are testing, the plan has the same skeleton.
Part of the plan
What to write
Independent variable
Temperature, pH or substrate concentration — one only, with at least five values across a sensible range
Dependent variable
Rate, measured as product formed or substrate lost, with units stated
Control variables
The other two factors, plus enzyme concentration, volume and total time
Apparatus choice
Follows from the dependent variable: gas syringe or measuring cylinder for oxygen, colorimeter for a colour change
Repeats
At least three at each value, then take a mean and ignore obvious anomalies
Method 1: catalase and hydrogen peroxide (product appearing)
Hydrogen peroxide is a toxic by-product of metabolism, so cells must break it down quickly. Catalase does it, splitting hydrogen peroxide into water and oxygen. The oxygen is a gas, which makes it easy to collect and measure.
The reaction
hydrogen peroxide → water + oxygen (catalysed by catalase)
To test temperature, stand the flask in a water bath and let it reach that temperature before adding the hydrogen peroxide.
🧩 Running it properly
Prepare identical potato pieces. Same number, same size, same mass every time — this keeps enzyme amount constant.
Set the temperature. Stand the flask in a water bath, with the water level above the level of liquid inside the flask so heating is even.
Let it equilibrate. Give the contents a few minutes to reach the bath temperature before you start.
Add the hydrogen peroxide, bung it, start the clock in that order. Gas escaping before the bung is in is a common source of error.
Swirl gently to keep the mixture evenly mixed and evenly heated.
Record the volume of oxygen at fixed time intervals, then repeat the whole thing at each temperature.
Method 2: amylase and starch (substrate disappearing)
Sometimes there is no gas to collect, so you follow the substrate instead. Amylase hydrolyses starch into maltose and glucose, and starch is easy to test for: a drop of iodine solution turns blue-black if starch is present, and stays yellow-brown if it is not.
Mix amylase and starch, then take a small sample at regular intervals.
Add each sample to a drop of iodine solution on a spotting tile.
The time at which the iodine stops turning blue-black is the time all the starch has been broken down.
Repeat with different starch concentrations and compare. The same set-up also works for changing pH or temperature.
Making the concentrations: a serial dilution is the neat way to do it. Take 1 cm3 of your stock solution, add 9 cm3 of water, mix, then take 1 cm3 of that and repeat. Each step is ten times weaker than the one before.
Method 3: using a colorimeter
A colorimeter measures how much light a solution absorbs or transmits, so it can follow any enzyme reaction involving a colour change. As the starch–iodine colour fades, absorbance falls and transmission rises.
Calibrate first — here, a weak iodine solution is set as 100% transmission.
Prepare a range of known starch concentrations by serial dilution.
Select the red filter and read the percentage absorbance or transmission for each.
Plot a calibration graph of concentration against absorbance, then read unknown samples off it.
Worked examples
WORKED EXAMPLE
Explain the shape of the graph of rate against temperature between 5°C and 55°C. [4]
Step 1: split the graph in two
Rise up to the optimum, then a sharp fall after it.
Step 2: explain the rise
More kinetic energy → faster movement → more successful collisions → more complexesStep 3: explain the fall
Weak bonds break, the active site changes shape, substrate no longer complementary.
Rate rises to the optimum because of collisions, then falls because the enzyme denaturesfour marks usually means two ideas each side — do not spend them all on the rise
WORKED EXAMPLE
A student investigates pH using catalase. Give the independent variable, the dependent variable and two control variables. [4]
Step 1: what is being changedIV = pH of the buffer solutionStep 2: what is being measuredDV = volume of oxygen collected in cm3 in a fixed timeStep 3: what must stay the same
Temperature, and the mass or surface area of potato used.
IV pH, DV volume of oxygen per unit time, controls temperature and enzyme amountsubstrate concentration and total volume are also good control answers
WORKED EXAMPLE
A reaction has reached its plateau on a graph of rate against substrate concentration. Suggest how to increase the rate further, and explain why. [2]
Step 1: name what is limiting the rate
All active sites are occupied, so the enzyme is the limit — not the substrate.
Step 2: fix that limitmore enzyme = more active sites availableIncrease the enzyme concentration“add more substrate” scores nothing here — that is exactly what has stopped working
💡 Exam tip
Learn all three sketch graphs as shapes, including the axes labels. You may be asked to draw a second curve for changed conditions on the same axes.
For temperature, always give two explanations — collisions below the optimum, denaturation above it.
Say “more successful collisions”, not just “more collisions”. The extra word is often the mark.
When describing a graph, quote a value: “the rate peaks at about 35°C” beats “it peaks in the middle”.
Give the dependent variable units in any plan question. Volume of oxygen in cm3 per minute, for example.
If asked why potato is used, the answer is that it is a convenient source of catalase — the potato itself is not the point.
⚠ Common mix-up
Saying enzymes are denatured by low temperature. They are not. Cold slows them down and the effect is reversible.
Drawing a symmetrical temperature curve. The fall after the optimum is much steeper than the rise before it.
Letting the substrate-concentration curve drop after the plateau. It levels off and stays level.
Assuming every optimum pH is 7. Pepsin is around 2, and some bacterial enzymes prefer 9 or 10.
Treating pH as a linear scale. One unit is a tenfold change in hydrogen ion concentration.
Explaining the plateau with “the enzyme is denatured”. Nothing is denatured at saturation — the active sites are simply all busy.
Forgetting to let the flask reach the water bath temperature before adding the substrate. It is a favourite “improve the method” answer.
Up next: Enzyme Reaction Rates (Skills) — turning your raw readings into an actual number, drawing tangents and calculating the initial rate.
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