Enzymes

• Function follows shape, and shape is held by weak bonds. Every factor in this topic works either by changing how often substrate meets an active site, or by changing the active site itself.
• Complementary, not identical. Lower activation energy, same overall change. Km is a substrate concentration at ½Vmax: low Km, high affinity.

• 3.1.1 I can state — state that enzymes are globular proteins that catalyse reactions inside cells (intracellular enzymes) or are secreted to catalyse reactions outside cells (extracellular enzymes)
• 3.1.2 I can explain — explain the mode of action of enzymes in terms of an active site, enzyme–substrate complex, lowering of activation energy and enzyme specificity, including the lock-and-key hypothesis and the induced-fit hypothesis
• 3.1.3 I can investigate — investigate the progress of enzyme-catalysed reactions by measuring rates of formation of products using catalase and rates of disappearance of substrate using amylase
• 3.1.4 I can outline — outline the use of a colorimeter for measuring the progress of enzyme-catalysed reactions that involve colour changes
• 3.2.1 I can investigate — investigate and explain the effects of the following factors on the rate of enzyme-catalysed reactions: • temperature • pH (using buffer solutions) • enzyme concentration • substrate concentration • inhibitor concentration
• 3.2.2 I can explain — explain that the maximum rate of reaction (Vₘₐₓ) is used to derive the Michaelis–Menten constant (Kₘ), which is used to compare the affinity of different enzymes for their substrates
• 3.2.3 I can explain — explain the effects of reversible inhibitors, both competitive and non-competitive, on enzyme activity
• 3.2.4 I can investigate — investigate the difference in activity between an enzyme immobilised in alginate and the same enzyme free in solution, and state the advantages of using immobilised enzymes

Why collisions matter for the rest of the chapter. An ES complex forms only when substrate meets a working active site. So every factor in 3.2.1 acts in one of two ways: it changes how often successful collisions happen (temperature below the optimum, enzyme and substrate concentration), or it changes the number of active sites that can bind (temperature above the optimum, pH, inhibitors).

• “The substrate and the active site are identical in shape.” Correct The active site is complementary to the substrate: the substrate fits into it, as a hand fits into a glove, and R groups in the site form temporary bonds with it. Use the word complementary every time.
• “The enzyme gives the substrate the energy it needs to react.” Correct An enzyme supplies no energy. It lowers the activation energy, so more substrate molecules already have enough energy to react. The overall energy change of the reaction is the same with or without it.
• “At 5 °C the enzyme is denatured.” Correct At low temperature the molecules have little kinetic energy and collide less often, so the rate is low, but the active site keeps its shape. Warm it and activity returns. Denaturation is a change in the tertiary structure caused by heat or extreme pH.
• “The rate levels off at high substrate concentration because the substrate runs out.” Correct On a rate–substrate concentration graph each point has plenty of substrate. The rate levels off because the active sites are saturated; enzyme concentration is now the limiting factor. Substrate being used up explains why a product–time curve levels off — a different graph.
• “Km is half of Vmax, and a high Km means a high affinity.” Correct Km is a substrate concentration: the one at which the rate is half of Vmax. A low Km means a high affinity.
• “A competitive inhibitor lowers Vmax.” Correct Raise the substrate concentration far enough and substrate out-competes the inhibitor, so the same Vmax is approached; Km rises. It is the non-competitive inhibitor that lowers Vmax, leaving Km unchanged.
• “The control was keeping the temperature the same.” Correct Keeping temperature the same is standardising a variable. A control is a separate tube — boiled and cooled enzyme, or water in place of enzyme — that shows the change is caused by the enzyme.
• “The substrate is the same shape as the active site.” Repair The active site is complementary in shape to the substrate: the two fit together, the substrate sitting into the site. “The same shape” would mean two copies of one shape, which could not fit into each other.
• “At high temperature the enzyme dies.” Repair An enzyme is a protein, not a living thing. It is denatured: hydrogen and ionic bonds in its tertiary structure break and the active site changes shape.
• “At low temperature the enzyme is denatured.” Repair At low temperature molecules have little kinetic energy and collide less often. The enzyme is inactive, but its shape is intact and warming it restores activity.
• “Enzymes provide the energy for the reaction.” Repair Enzymes lower the activation energy; they supply no energy, and the overall energy change of the reaction is unchanged.
• “The rate levels off because the substrate runs out.” (about a rate–substrate concentration graph) Repair On that graph every point has plenty of substrate; the rate levels off because the active sites are saturated. Substrate running out explains why a product–time curve levels off.
• “A high Km means a high affinity.” Repair A low Km means a high affinity: half the maximum rate at a low substrate concentration.
• “Km is half of Vmax.” Repair Km is a substrate concentration: the one at which the rate is half of Vmax. Its unit is a concentration unit.
• “Vmax is the highest rate in the table.” Repair Vmax is approached, not reached. Read it from the level the curve is flattening towards; taking the last reading makes both Vmax and Km too low.
• “A competitive inhibitor lowers Vmax.” Repair At high substrate concentration the substrate out-competes it, so Vmax is still approached; the apparent Km rises. It is the non-competitive inhibitor that lowers Vmax.
• “A non-competitive inhibitor blocks the active site.” Repair It binds elsewhere on the enzyme and changes the shape of the active site.
• “Buffer is added to keep the reaction going.” Repair A buffer keeps the pH constant at the chosen value throughout the reaction.
• “The control is keeping the temperature the same.” Repair Keeping temperature the same is standardising a variable. A control is a separate tube, e.g. boiled and cooled enzyme, that shows the enzyme causes the change.
• “The mean rate over the first 30 s is the initial rate.” Repair The initial rate is the gradient of the tangent at t = 0. The first-interval mean includes the slower rates that follow and underestimates it.
• “Immobilised enzymes work faster.” Repair They are often slower at first, because substrate must diffuse into the beads. Their advantages are reuse, a product free of enzyme, greater stability and a continuous process.
• “Lock-and-key and induced fit are the same idea.” Repair In induced fit the active site changes shape slightly as the substrate binds, which strains bonds in the substrate. In lock-and-key the site is rigid.

• Read the command word before you decide how much to write. This syllabus has seventeen of them: assess, calculate, comment, compare, contrast, define, describe, determine, discuss, explain, give, identify, outline, predict, sketch, state and suggest. State, give and identify want a fact and nothing more. Define wants a precise meaning. Outline wants the main points only; describe wants the points or the features in full — and when you describe a graph, the trend with figures quoted from it. Explain wants the reasons and the mechanism — a describe-level answer to an explain question is incomplete however well written it is. Compare wants similarities and differences, each stated for both things side by side; contrast wants differences only. Discuss wants the issue written about in depth, in a structured way; assess wants an informed judgement. Suggest asks you to apply what you know to a situation where there is a range of valid responses, making proposals or putting forward considerations, so any sound biological reasoning is creditable.
• Interleave with the chapters that use this one. In topics 12 and 13, re-answer “why does temperature change the rate?” for the enzymes of respiration and photosynthesis. In topic 14, re-answer “what makes an enzyme specific?” when enzymes are used to measure blood glucose (14.1.11). In topic 19, re-answer “what does denaturation do to an enzyme?” for the heat-stable polymerase used in PCR. Recalling a topic inside a new context is worth more than another pass over this chapter on its own; at A Level, Paper 4 assumes the whole of the AS content, so nothing here is ever finished with.

• Cambridge International AS & A Level Biology 9700 has five components. Topic 3 is AS Level content, so it is examined in Papers 1, 2 and 3. AS Level content: examined in Paper 1 (multiple choice), Paper 2 (AS structured) and, as practical context, Paper 3. Assumed knowledge for Papers 4 and 5. AS Level candidates take Papers 1, 2 and 3; A Level candidates take all five, either staged over two years (Papers 1–3 in year one, Papers 4 and 5 in year two) or together in one series. Examinations are available in the June and November series, and in March in India.
• Across both the AS Level and the A Level the assessment objectives are weighted AO1 40% (knowledge and understanding), AO2 40% (handling, applying and evaluating information) and AO3 20% (experimental skills and investigations). AS candidates are graded a–e; A Level candidates A*–E. There is no data booklet in Biology. At A Level, the statistical formulae (Hardy–Weinberg, the Lincoln index, Simpson’s index, standard deviation, standard error, 95% confidence intervals, the χ² test, the t-test, and Pearson’s and Spearman’s correlation) are printed in a question when it needs them, and so are the tables of critical values; degrees of freedom you must work out yourself. Everything else — magnification, surface area to volume, RQ, Rf, rates — you must recall, and this chapter says which is which.
• A multiple-choice item on this topic can turn on a single distinction: complementary or identical, Vmax or Km, competitive or non-competitive, a control or a standardised variable (Paper 1). A structured question may ask you to state what enzymes are in the syllabus's words, describe a rate graph with figures quoted, explain a change in rate through collisions or the active site, compare the two hypotheses or the two inhibitors, or calculate a rate (Paper 2).
• Product–time curves and the tangent at t = 0; rate against temperature, pH or concentration; a rate–substrate curve from which to read Vmax and Km; curves with and without an inhibitor; a colorimeter calibration line. The numerical skills: rate = 1 / time, a gradient with its unit, halving Vmax, a percentage of an uninhibited rate, reading a value from a line.
• Oxygen collected from hydrogen peroxide by catalase, or starch disappearing with amylase and iodine; one of the five factors as the independent variable, changed by water baths, buffer solutions or proportional dilution; volume of oxygen or time to end-point as the dependent variable; temperature, pH and volumes standardised; boiled enzyme as the control; oxygen lost before sealing, or an end-point known only to the sampling interval, as the main error. Immobilised enzyme beads are a context too.
• Read the command word before you decide how much to write. This syllabus has seventeen of them: assess, calculate, comment, compare, contrast, define, describe, determine, discuss, explain, give, identify, outline, predict, sketch, state and suggest. State, give and identify want a fact and nothing more. Define wants a precise meaning. Outline wants the main points only; describe wants the points or the features in full — and when you describe a graph, the trend with figures quoted from it. Explain wants the reasons and the mechanism — a describe-level answer to an explain question is incomplete however well written it is. Compare wants similarities and differences, each stated for both things side by side; contrast wants differences only. Discuss wants the issue written about in depth, in a structured way; assess wants an informed judgement. Suggest asks you to apply what you know to a situation where there is a range of valid responses, making proposals or putting forward considerations, so any sound biological reasoning is creditable.