What you'll learn
Enzymes and metabolism is one of the most heavily examined topics in CAPE Biology Unit 1, because enzymes control every reaction in every organism and because the topic lends itself so readily to graph interpretation and experimental design. At CAPE level you are expected to go beyond the lock and key model to the induced fit model, to distinguish competitive from non-competitive inhibition and recognise each from a graph, to understand the meaning of Vmax and Km, and to explain the industrial use of immobilised enzymes. By the end of this topic you should be able to explain enzyme action and specificity, describe the effect of every factor on rate with the underlying reason, distinguish the types of inhibition, interpret rate graphs confidently, and design a valid investigation.
Key terms and definitions
Enzyme — a biological catalyst, a globular protein that increases the rate of a reaction without being changed
Catalyst — a substance that increases reaction rate without being used up
Active site — the region of an enzyme with a shape complementary to its substrate
Substrate — the molecule on which an enzyme acts
Enzyme–substrate complex — the temporary structure formed when substrate binds to the active site
Activation energy — the minimum energy required for a reaction to proceed
Induced fit model — the model in which the active site changes shape slightly to mould around the substrate
Denaturation — loss of the tertiary structure and therefore of the active site's shape
Competitive inhibitor — a molecule similar in shape to the substrate that binds to the active site
Non-competitive inhibitor — a molecule binding away from the active site, altering its shape
Allosteric site — a site on an enzyme other than the active site to which a regulator binds
Vmax — the maximum rate of reaction when all active sites are saturated
Km — the substrate concentration at which the rate is half of Vmax, a measure of affinity
Immobilised enzyme — an enzyme attached to or trapped in an inert support
Core concepts
How enzymes work
Every chemical reaction requires an input of energy to begin, called the activation energy. Enzymes work by lowering the activation energy required, providing an alternative route by which the reaction can proceed. A far greater proportion of substrate molecules therefore have sufficient energy to react at the temperature of the organism, so the rate increases enormously.
Enzymes do not change the equilibrium position of a reaction or the overall energy change. They only allow it to be reached faster, and they are not consumed, so a single enzyme molecule catalyses many reactions in succession.
Enzymes may be intracellular, acting within the cell that made them, or extracellular, secreted to act outside, as digestive enzymes do.
Specificity and the two models
Each enzyme catalyses only one reaction or one class of reaction, because the shape of the active site is complementary to only one substrate. That shape is determined by the tertiary structure, which is itself determined by the primary sequence, and ultimately by the gene.
The lock and key model proposes that the substrate fits an active site of precisely complementary shape, like a key in a lock. It explains specificity but treats the enzyme as rigid.
The induced fit model, which is the accepted model at this level, proposes that the active site is not an exact fit initially. As the substrate approaches, the active site changes shape slightly to mould more closely around it. This change places strain on the substrate's bonds, weakening them and further lowering the activation energy.
The induced fit model is preferred because it explains how enzymes actively lower activation energy rather than merely holding the substrate, and because it accounts for enzymes that act on a range of similar substrates.
Temperature
As temperature increases from low values, the rate rises because both enzyme and substrate molecules gain kinetic energy, move faster and collide more frequently, so more enzyme–substrate complexes form per unit time.
The rate reaches a maximum at the optimum temperature, which for most human enzymes is around 40 degrees Celsius, though enzymes from thermophilic bacteria have far higher optima.
Above the optimum the rate falls sharply. The increased vibration breaks the hydrogen bonds and ionic bonds maintaining the tertiary structure, so the active site changes shape and is no longer complementary to the substrate. Fewer enzyme–substrate complexes can form, and the enzyme is denatured.
Denaturation is permanent, because the bonds do not reform in the correct arrangement on cooling. This distinguishes the effect of high temperature from that of low temperature, where activity resumes on warming because the enzyme was merely inactive, not denatured. That distinction is frequently examined.
The temperature coefficient Q10 expresses the effect quantitatively: it is the rate at a given temperature divided by the rate ten degrees lower, and for enzyme-controlled reactions below the optimum it is approximately 2, meaning the rate roughly doubles for each 10 degree rise.
pH
Each enzyme has an optimum pH, and the rate falls either side of it. Pepsin in the stomach has an optimum near pH 2, while trypsin in the small intestine works best near pH 8.
A change in pH alters the concentration of hydrogen ions, which affects the charges on the R groups of amino acids in the enzyme. This disrupts the ionic bonds and hydrogen bonds holding the tertiary structure, so the active site changes shape and substrate binding is reduced.
Small deviations cause a reversible loss of activity, but extremes cause permanent denaturation.
Substrate and enzyme concentration
At low substrate concentration, the rate is directly proportional to substrate concentration, because there are many free active sites and substrate availability is the limiting factor.
As substrate concentration rises, the rate levels off. At this point every active site is occupied as soon as it becomes free, so the enzyme is saturated and the enzyme concentration has become the limiting factor. The maximum rate reached is Vmax.
Km is the substrate concentration giving half of Vmax, and it measures the affinity of the enzyme for its substrate. A low Km indicates high affinity, because only a small substrate concentration is needed to achieve half-maximal rate.
Increasing enzyme concentration increases the rate proportionally, provided substrate is in excess, because more active sites are available.
Inhibition
A competitive inhibitor has a shape similar to the substrate and binds to the active site, physically preventing the substrate from binding. The two compete for the same site, so the effect depends on their relative concentrations.
Increasing the substrate concentration therefore reduces the effect of a competitive inhibitor, and Vmax is eventually reached — it just takes more substrate. On a graph, a competitive inhibitor raises Km but leaves Vmax unchanged.
A non-competitive inhibitor binds at a site other than the active site, often an allosteric site. This alters the tertiary structure of the enzyme so that the active site changes shape and the substrate no longer fits.
Because the inhibitor is not competing for the active site, increasing substrate concentration does not overcome it. On a graph, a non-competitive inhibitor lowers Vmax while Km is unchanged.
Distinguishing the two from a graph is a standard question, and the reliable test is what happens at high substrate concentration: if the inhibited curve eventually reaches the uninhibited Vmax, the inhibition is competitive.
End-product inhibition is an important regulatory application. The product of a metabolic pathway acts as a non-competitive inhibitor of an enzyme earlier in that pathway, so that the pathway shuts down when sufficient product has accumulated. This is negative feedback at the molecular level and prevents wasteful overproduction.
Cofactors, coenzymes and prosthetic groups
Many enzymes require an additional non-protein component.
A cofactor is any such component. Inorganic cofactors are ions such as chloride ions, which are needed by amylase.
A coenzyme is an organic cofactor that binds temporarily, often derived from a vitamin, and typically carries chemical groups or electrons between enzymes. NAD and coenzyme A in respiration are the examples you will meet.
A prosthetic group is a cofactor bound permanently to the enzyme, such as the haem group in catalase.
Immobilised enzymes
Immobilised enzymes are attached to or trapped within an inert support such as alginate beads, a membrane or a gel.
The advantages are examinable and substantial. The enzyme can be recovered and reused, greatly reducing cost. The product is not contaminated with enzyme, so purification is simpler and the product is safe for food and pharmaceutical use. Immobilised enzymes are more stable to changes in temperature and pH, because the support holds the tertiary structure in place. The reaction can be run continuously rather than in batches, and stopped readily by removing the beads.
The disadvantage is that the rate is usually somewhat lower, because the substrate must diffuse to the immobilised enzyme and the support may partially obstruct the active site, and the initial setup cost is higher.
Applications include lactase immobilised on beads to produce lactose-free milk, and glucose isomerase to convert glucose to the sweeter fructose for food manufacture.
Designing an investigation
Enzyme practicals recur, and the design elements carry marks.
The independent variable is the factor being changed — temperature, pH, substrate concentration or enzyme concentration. The dependent variable is the rate, measured for example as the volume of oxygen produced by catalase per unit time, the time for a starch solution to lose its blue-black colour with iodine, or the time for a suspension to clear.
Control variables include all the factors not being investigated, and specifically the volume and concentration of enzyme and substrate, the pH maintained with a buffer solution, and the temperature maintained with a water bath.
Rate is calculated as one divided by the time where a time-to-completion method is used, since a shorter time means a faster reaction.
Reliability is improved by repeating each condition and calculating a mean, and by identifying and excluding anomalous results before averaging.
Worked examples
Example 1: Distinguishing inhibitors from a graph (5 marks)
Two inhibitors are tested on the same enzyme. With inhibitor A, the curve of rate against substrate concentration rises more slowly but eventually reaches the same maximum rate as the uninhibited enzyme. With inhibitor B, the curve levels off at a lower maximum rate. Identify each and explain.
Inhibitor A is competitive. It has a shape similar to the substrate and binds to the active site, competing with the substrate. Because the two compete for the same site, increasing the substrate concentration increases the proportion of active sites occupied by substrate rather than inhibitor, so the original maximum rate is eventually reached. Km is increased but Vmax is unchanged.
Inhibitor B is non-competitive. It binds at a site other than the active site, altering the tertiary structure so that the active site changes shape and the substrate can no longer bind. Since it does not compete for the active site, increasing substrate concentration cannot displace it, so a proportion of enzyme molecules remain permanently inactive and Vmax is reduced.
Example 2: Explaining a temperature curve (5 marks)
Explain the shape of a graph showing enzyme activity against temperature from 0 to 60 degrees Celsius.
From 0 degrees upwards, the rate increases because both enzyme and substrate molecules gain kinetic energy and move faster. Collisions between them become more frequent, so more enzyme–substrate complexes form per unit time.
The rate reaches a peak at the optimum temperature, where the rate of successful collisions is greatest without significant damage to the enzyme.
Above the optimum the rate falls steeply. The increased vibration of the molecule breaks the hydrogen bonds and ionic bonds maintaining the tertiary structure, so the active site changes shape and is no longer complementary to the substrate. Fewer enzyme–substrate complexes form.
At high temperatures the enzyme is fully denatured and the rate falls to zero. This is irreversible, whereas the low activity at 0 degrees is reversible on warming, because the enzyme there is inactive rather than denatured.
Example 3: Explaining end-product inhibition (4 marks)
Explain how end-product inhibition regulates a metabolic pathway.
In a metabolic pathway, a series of enzymes converts an initial substrate into a final product through intermediates.
The final product acts as a non-competitive inhibitor of an enzyme near the start of the pathway, binding to an allosteric site on that enzyme. This alters the enzyme's tertiary structure so that its active site changes shape and can no longer bind its substrate.
As the concentration of the end product rises, more of the early enzyme is inhibited and the pathway slows. As the product is used up, inhibition is relieved and the pathway resumes.
This is a form of negative feedback, ensuring the product is made only as fast as it is needed and preventing the wasteful accumulation of intermediates and product.
Common mistakes and how to avoid them
The most frequently penalised error is writing that high temperature kills the enzyme. An enzyme is a molecule and is denatured, not killed.
Students often state that the active site is destroyed. It changes shape, and the distinction matters to examiners.
Another common slip is claiming that enzymes are used up in the reaction, or that they change the equilibrium position. They are unchanged and reusable, and they affect only the rate.
Many candidates say that low temperature denatures enzymes. Low temperature reduces kinetic energy and activity reversibly; only high temperature and extreme pH denature.
In inhibition questions, answers frequently identify the type without explaining the effect on Vmax and Km. Both are usually required.
Finally, candidates often omit the buffer from an experimental design. If pH is not the variable under investigation, it must be controlled with a buffer solution.
Exam technique for "Enzymes and metabolism"
For any rate explanation, build the answer in a chain: what happens to the molecules, what happens to collisions or complexes, and therefore what happens to the rate.
Use the phrase enzyme–substrate complex explicitly. It appears in most mark schemes and is a reliable mark.
When interpreting a graph, describe the shape in sections and give the limiting factor for each. Rising means substrate is limiting; level means the enzyme is saturated.
For inhibition questions, state the binding site, the effect on the active site, whether increasing substrate overcomes it, and the effect on Vmax and Km — four separate points.
In experimental design, name the independent, dependent and at least three control variables, and say how each control is achieved, such as a water bath for temperature and a buffer for pH.
Quick revision summary
Enzymes are globular proteins that lower activation energy, are not used up, and do not alter the equilibrium position. Specificity arises from an active site whose shape is determined by tertiary structure; the induced fit model, in which the active site moulds around the substrate and strains its bonds, is preferred to the lock and key model. Rate rises with temperature as collisions increase, peaks at the optimum, then falls as hydrogen and ionic bonds break and the enzyme denatures irreversibly, while cold merely inactivates reversibly. pH affects the charges on R groups and so the tertiary structure. Rate rises with substrate concentration until active sites are saturated at Vmax, with Km being the substrate concentration giving half Vmax and indicating affinity. Competitive inhibitors bind the active site, raise Km and leave Vmax unchanged since extra substrate overcomes them; non-competitive inhibitors bind elsewhere, alter the active site and lower Vmax. End-product inhibition regulates pathways by negative feedback. Cofactors, coenzymes such as NAD, and prosthetic groups assist many enzymes. Immobilised enzymes can be reused, leave an uncontaminated product and resist temperature and pH change, at some cost in rate.