Enzyme activity vs. temperature, pH, substrate concentration and inhibitors

BiologyCell BiologyAges 15–16Virtual lab

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Use with my class

The enzyme (with an active site) and substrate molecules move around and collide; when a substrate molecule fits the active site it is converted into product. Students drag temperature, pH and substrate concentration, add a competitive or non-competitive inhibitor, and watch how the rate of product formation changes; graphs of activity versus temperature and pH show the current working point, and the enzyme denatures when overheated.

Lesson: Enzymes: mechanism of action and factors affecting enzyme activity

What it shows

Enzymes are proteins whose active site binds only substrates of a matching shape. The simulation models the rate as v = Vmax·f(T)·f(pH)·[S]/(Km + [S]). Below the optimum of about 37 °C, a 10 °C rise roughly doubles the rate; above 55 °C the protein denatures and does not recover on cooling. Each enzyme has its own optimum pH: pepsin works in stomach acid, trypsin in the alkaline small intestine. A competitive inhibitor raises the apparent Km and can be outcompeted by more substrate, whereas a non-competitive inhibitor lowers Vmax whatever the substrate concentration.

How to use

Pick an Enzyme, start at body temperature and its optimum pH, and note the measured rate of product formation. Change one factor at a time: raise the Temperature past the optimum, move the pH away from the optimum, then return each slider to see what recovers. Next choose an Inhibitor, note the drop in rate, and press +20 substrate several times. Press Reset after the enzyme has denatured.

Parameters you can change

Enzyme Salivary amylase (optimum pH 7), Gastric pepsin (optimum pH 2), Small intestine trypsin (optimum pH 8)
Temperature 0–80 °C
pH 1–13
Substrate concentration (number of molecules) 10–120
Inhibitor None, Competitive inhibition (competes for the active site), Non-competitive inhibition (binds elsewhere)

Questions to explore

Why does pepsin lose almost all its activity when the pH is set to 8?
After heating the enzyme to 60 °C and cooling it back to 37 °C, does its activity return? Why?
How does adding more substrate reveal whether an inhibitor is competitive or non-competitive?

Common questions

What is the active site of an enzyme, and why does each enzyme work on so few substrates?

The active site is a small pocket on the enzyme whose shape and chemical groups fit only certain substrate molecules, which is why each enzyme is specific. The substrate binds there to form an enzyme–substrate complex, the reaction takes place and the product leaves, so the unchanged enzyme can repeat the cycle many times.

Does cold destroy enzymes, or does it only slow them down?

Cold normally only slows enzymes down and does not destroy them, because the protein usually keeps its shape and activity returns on warming. At low temperature, enzyme and substrate molecules move more slowly and collide less often, so fewer complexes form each second, which is why chilled food spoils more slowly.

Why does the reaction rate stop rising when more substrate is added?

The rate levels off because every enzyme molecule is already occupied, so extra substrate must wait for a free active site, and this maximum rate is called Vmax. In v = Vmax·[S]/(Km + [S]), Km is the substrate concentration at which the rate reaches half of Vmax, so a small Km means the enzyme saturates at low substrate concentration.

Teaching ideas

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