Temperature, pH, the amounts of enzyme and substrate, and inhibitors all change how fast an enzyme works. Each effect comes back to two things: how often substrate meets a free active site, and whether that active site still has the right shape.
Raising the temperature increases kinetic energy and successful collisions until the optimum; above it, hydrogen and ionic bonds break, the active site loses its shape and the enzyme is denatured. Each enzyme has an optimum pH, kept constant in experiments with buffer solutions; other pH values alter the charges on R groups in the active site. Rate rises with enzyme concentration while substrate is in excess, and with substrate concentration until every active site is occupied and the rate reaches Vmax. The Michaelis-Menten constant Km is the substrate concentration giving ½Vmax: a low Km means high affinity. A competitive inhibitor competes for the active site (Vmax unchanged, Km raised); a non-competitive inhibitor binds elsewhere and changes the active site (Vmax lowered). Enzymes immobilised in alginate can be reused, do not contaminate the product and are more stable.
Fig. 1: rate against substrate concentration with no inhibitor, a competitive inhibitor and a non-competitive inhibitor
Section 01
Temperature: faster, then denatured.
Drag the temperature slider for the human enzyme (optimum 37 °C), then switch to the Antarctic fish enzyme and the thermophile enzyme (optimum 75 °C). Watch the active site distort as you pass each optimum.
Below the optimum: more heat means more kinetic energy, so substrate and enzyme collide more often and more collisions exceed the activation energy. The rate roughly doubles for each 10 °C rise (temperature coefficient, Q10, about 2).
At the optimum: the rate is highest; for most human enzymes this is close to 37 °C.
Above the optimum: vibrations break hydrogen bonds and ionic bonds holding the tertiary structure, so the active site changes shape, substrate no longer fits and fewer enzyme-substrate complexes form.
Denaturation is permanent; cooling a denatured enzyme does not restore it. Enzymes cooled below the optimum are only less active, not denatured.
Examiner trap
Do not write that enzymes are killed: they are not alive. Say the enzyme is denatured and explain it: bonds in the tertiary structure break, the active site loses its complementary shape, and fewer enzyme-substrate complexes form.
Section 02
pH and the charges in the active site.
Choose pepsin, salivary amylase, trypsin or arginase and move the pH slider. Note each optimum (pH 2, 6.8, 8 and 9.8 in the lab) and watch the charges on the active site change as H+ or OH− ions are added.
Charged R groups: amino acids in and around the active site carry charges that help bind the substrate.
Away from the optimum: extra H+ or OH− ions change those charges, so the substrate binds less well, and ionic and hydrogen bonds in the tertiary structure are disrupted.
Extreme pH denatures the enzyme; small shifts are often reversible.
Buffer solutions keep the pH constant in each tube, because the reaction itself, or the added substrate, could change it. Use a different buffer for each pH tested.
Stage 1 · Learn
Check what the sim just showed you
Four quick checks. Each correct answer earns XP and records this skill on your profile.
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In the temperature simulator, why does the rate of the human enzyme fall steeply above about 45 °C?
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Which enzyme in the simulator has the highest optimum temperature, and why?
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Pepsin has an optimum near pH 2. What happens to it in the small intestine at about pH 8?
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Why are buffer solutions used when investigating the effect of pH on an enzyme?
Section 03
Concentration, Vmax and inhibitors.
With no inhibitor, slide [S] up and watch the rate level off at Vmax; read Km = 4 mmol. Double [E]. Then add 1 mmol of competitive inhibitor (apparent Km doubles to 8 mmol) and of non-competitive inhibitor (Vmax halves).
Factor increased
Effect on rate
Reason
enzyme concentration
rises in proportion, if substrate is in excess
more active sites available; Vmax rises
substrate concentration
rises, then levels off at Vmax
at high [S] all active sites are occupied, so enzyme concentration becomes limiting
competitive inhibitor
falls, but can be overcome by more substrate
similar shape to substrate; binds the active site and blocks it
non-competitive inhibitor
falls, and more substrate does not restore it
binds elsewhere on the enzyme and changes the shape of the active site
Km: the substrate concentration at which the rate is half of Vmax. Find Vmax from the plateau, halve it, read across to the curve and down to the axis.
Affinity: an enzyme with a low Km reaches half its maximum rate at a low substrate concentration, so it has a high affinity for its substrate.
Reversible inhibitors bind by weak, temporary bonds and can leave the enzyme. Competitive: Vmax unchanged, apparent Km raised. Non-competitive: Vmax lowered, Km unchanged. The more inhibitor, the greater the effect.
Examiner trap
A competitive inhibitor does not lower Vmax: at a very high substrate concentration the substrate out-competes it. Only a non-competitive inhibitor lowers Vmax. And Km is a substrate concentration, read on the x-axis, not a rate.
Section 04
Immobilised in alginate.
Run several batches through the free enzyme and the alginate beads at 37 °C, then raise the temperature above 55 °C and compare the yields.
Making beads: mix the enzyme with sodium alginate solution and drip it into calcium chloride solution; insoluble calcium alginate beads form with the enzyme trapped inside.
Comparing activity: pack beads in a column or syringe barrel, run substrate through, and test the product (for example, glucose test strips for lactase acting on lactose). Compare with the same quantity of free enzyme in solution, and use beads made without enzyme as a control.
Advantages: the enzyme can be reused; the product is not contaminated with enzyme; the enzyme is more stable over a wider range of temperature and pH; a continuous process is possible.
Drawback: the rate can be slightly lower, because substrate must diffuse into the beads to reach the active sites.
Stage 2 · Exam
Exam-style questions
Unlocks once the four checks above are done. Worth more XP, written to AS Paper 1 and Paper 2 standard.
Finish the four checks above to unlock the exam questions
Exam style+20 XP
Enzyme A has a Km of 0.5 mmol dm−3 and enzyme B a Km of 5.0 mmol dm−3 for the same substrate. Which statement is correct?
Exam style+20 XP
Adding a large excess of substrate restores the rate of an inhibited enzyme to its uninhibited Vmax. What type of inhibitor is present, and how does it act?
Exam style+20 XP
Which change is seen when a non-competitive inhibitor is added to an enzyme reaction?
Exam style+20 XP
Which is an advantage of using lactase immobilised in alginate beads rather than free lactase to make lactose-free milk?
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Factors that affect enzyme action, mastered.
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