Enzymes¶
Part of Module 2: Foundations in biology.
Enzymes are globular proteins that act as biological catalysts. Each enzyme's tertiary structure gives it an active site that fits one kind of substrate, so enzyme behaviour can be explained from protein structure. The rate of an enzyme-catalysed reaction depends on temperature, pH and the concentrations of substrate and enzyme, and inhibitors can lower it.
What You Need to Learn¶
Further detail: AS Biology A (H020) and A Level Biology A (H420).
Enzyme action covers how enzymes lower activation energy, where they act, and how the lock-and-key and induced-fit models describe substrate binding. Rate covers temperature, pH, and substrate and enzyme concentration, with Q10 as the measure of the temperature effect. Cofactors, coenzymes and prosthetic groups explain why some enzymes need a partner, and competitive and non-competitive inhibition explain how activity is controlled. All of it has to be applied to data: graphs, rate calculations and the results of practical work.
Enzyme Action¶
Enzymes are globular proteins with a complex, unique tertiary structure. As biological catalysts they speed up reactions without being used up, so a single enzyme molecule can catalyse many reactions in succession.
Every reaction needs a minimum amount of energy before it starts: the activation energy. It is needed to break the bonds in the reactants so that new bonds can form. Without an enzyme this energy usually comes from heat, and many biological reactions would be far too slow at body temperature. Enzymes lower the activation energy, so the reactions run at useful rates at the temperature of the organism.
Exam technique
To explain how an enzyme speeds up a reaction, give the sequence: the substrate binds to the active site, an enzyme–substrate complex forms, the activation energy is lowered, the products leave, and the enzyme is unchanged. Naming the active site and the activation energy is what turns "the enzyme speeds it up" into an explanation.
Intracellular and Extracellular Enzymes¶
Intracellular enzymes act inside the cells that make them. Catalase is an example. It breaks down hydrogen peroxide, a toxic by-product of metabolism, into water and oxygen before it can damage the cell.
Extracellular enzymes are secreted and act outside the cell that made them. Amylase is made in the salivary glands and the pancreas, and it breaks starch down into maltose in the mouth and the small intestine. Trypsin is made in the pancreas and acts in the small intestine, where it breaks proteins into smaller polypeptides.
Digestion needs extracellular enzymes because starch and protein molecules are too large to cross the cell surface membrane. They have to be broken down outside the cell first.
The Active Site and Enzyme–Substrate Complexes¶
An enzyme's tertiary structure fixes the shape and chemistry of its active site. The active site is specific to its substrate because it is complementary to it: not identical, but matched in shape and chemistry.
When the substrate binds, an enzyme–substrate complex forms. Temporary bonds, including hydrogen bonds and ionic interactions, hold the substrate to the R groups of the amino acids in the active site. These interactions lower the activation energy, either by straining the bonds in the substrate or by holding the reactants in the right orientation to react. The products are then released, and the enzyme is free to bind another substrate.
Lock-and-Key and Induced-Fit Models¶
Two models describe how the substrate binds.
The lock-and-key model treats the active site as a rigid shape that already matches one substrate. The substrate fits without the enzyme changing shape.
The induced-fit model treats the active site as flexible. It is still specific to its substrate, but it changes shape slightly as the substrate binds, which makes it more complementary. The change in shape also puts strain on the substrate's bonds, and that helps to lower the activation energy. Induced fit is the preferred model because it explains how the act of binding contributes to catalysis, which a rigid active site cannot.
The interactive below steps through both models.
Teacher insight
This interactive is a simplified 2D teaching model. Real enzymes are complex globular proteins with three-dimensional active sites, and the active site occupies only a small region of the whole enzyme rather than most of the protein. Use the diagram to understand specificity, complementary binding and induced fit, not as a literal picture of enzyme size or shape. Compare it with Globular proteins: 3D structures if you want a better sense of real protein folding.
Factors Affecting Rate¶
Four factors commonly change the rate of an enzyme-catalysed reaction: temperature, pH, substrate concentration and enzyme concentration. Temperature and pH act on the enzyme's structure as well as on how often molecules meet. The two concentrations change only how many molecules are available to meet.
The interactive below compares the four graph shapes. Use it to separate the two concentration graphs from the temperature and pH optima, and to keep saturation distinct from denaturation. Open full interactive.
Temperature¶
Every enzyme has an optimum temperature at which its rate is highest, and the value differs between enzymes. As the temperature rises towards the optimum, the molecules gain kinetic energy. Enzyme and substrate collide more often and with more energy, more enzyme–substrate complexes form, and the rate rises.
Above the optimum, the extra kinetic energy breaks the bonds that hold the enzyme's tertiary structure in shape. The active site changes shape, the substrate no longer fits, and the enzyme is denatured. Denaturation is irreversible. The rate falls steeply because denaturation outweighs the benefit of more frequent collisions.
Below the optimum, collisions are less frequent and less energetic, so the rate is low. The enzyme is not denatured, and its rate recovers when the temperature rises.
Exam technique
Enzymes are molecules and are not alive, so they are denatured and never "killed". Describe what changes: the bonds holding the tertiary structure break, so the active site changes shape. "The enzyme stops working" describes the result and not the cause.
Illustration: an enzyme with a high optimum
Optimum temperature reflects where an organism lives. Most human enzymes work best near 37 °C. DNA polymerase from Thermus aquaticus, a bacterium of hot springs, works best at roughly 70–80 °C and stays active after repeated heating to 95 °C, which is why it is used in PCR (see 6.1.3 Manipulating genomes).
Worked example: the temperature coefficient (Q10)
Q10 shows how much the rate changes for a 10 °C rise in temperature.
Q10 = R2 ÷ R1
R2 is the rate at the higher temperature and R1 is the rate at the lower one, and the two temperatures must be exactly 10 °C apart.
A reaction makes 5 products per minute at 20 °C and 10 products per minute at 30 °C.
Q10 = 10 ÷ 5 = 2
The rate doubles for every 10 °C rise, which is typical of biological reactions over a moderate range of temperature. Q10 is a ratio, so it has no units. A Q10 near 1 means temperature has little effect. A Q10 below 1 means the rate has fallen, as it does above the optimum when denaturation takes over.
pH¶
Every enzyme has an optimum pH, and this also differs between enzymes. In acidic conditions, excess H⁺ ions disrupt the ionic and hydrogen bonds in the tertiary structure. In alkaline conditions, OH⁻ ions do the same. In both cases the ions alter the charges on the R groups, so the active site changes shape, fewer enzyme–substrate complexes form, and the rate falls. A large enough shift in pH denatures the enzyme completely.
Illustration: optimum pH matches the environment
Pepsin acts in the stomach and works best at about pH 2, a pH that would denature many enzymes. Salivary amylase works best close to pH 7. Each enzyme's optimum is matched to the place where it works.
Substrate Concentration¶
At low substrate concentration, few substrate molecules are available and many active sites are empty. Adding substrate makes collisions with active sites more frequent, so the rate rises.
The relationship has a limit. When every active site is occupied at once, the enzyme is saturated and extra substrate has no effect, because enzyme concentration has become the limiting factor. The graph rises steeply and then plateaus at the maximum rate, Vmax.
Enzyme Concentration¶
More enzyme means more active sites. While substrate is in excess, the rate rises in proportion to enzyme concentration, so the graph is a straight line through the origin. Once enzyme molecules outnumber the substrate molecules available to fill them, substrate concentration becomes the limiting factor and the rate plateaus.
Cofactors, Coenzymes, and Prosthetic Groups¶
Some enzymes are active only with a non-protein partner called a cofactor. A cofactor binds to the enzyme and increases its activity.
| Cofactor | What it is | How it binds | Example |
|---|---|---|---|
| Inorganic cofactor | An inorganic ion | Binds to the enzyme and increases its activity | Chloride ions (Cl⁻) for amylase, which has reduced activity without them |
| Coenzyme | An organic molecule, often made from a vitamin | Temporarily, during the reaction | NAD, made from vitamin B3, carries hydrogen in respiration (see 5.2.2 Respiration) |
| Prosthetic group | A cofactor that is permanently and tightly bound to the enzyme | Permanently | Zinc ions (Zn²⁺) in carbonic anhydrase, which catalyses the conversion of carbon dioxide and water to carbonic acid in red blood cells |
Coenzymes often act as carriers, picking up atoms or groups from one reaction and donating them in another. Because they are not permanently bound, cells must keep recycling or replacing them.
The categories overlap. A prosthetic group is defined by permanent binding, so an ion such as Zn²⁺ is both an inorganic cofactor and a prosthetic group.
Enzyme Inhibitors¶
Inhibitors are molecules that bind to an enzyme and reduce its activity. Reversible inhibitors form weak bonds, such as hydrogen or ionic bonds, and can leave the enzyme. Irreversible inhibitors form strong covalent bonds and permanently disable it.
| Competitive | Non-competitive | |
|---|---|---|
| Where it binds | The active site | The allosteric site, away from the active site |
| How it reduces activity | Occupies the active site, so the substrate cannot bind | Changes the tertiary structure, so the active site is no longer complementary to the substrate |
| Effect of adding more substrate | Overcomes the inhibition | No effect |
| Vmax | Unchanged: still reached at a high enough substrate concentration | Lowered, however much substrate is added |
Competitive Inhibitors¶
A competitive inhibitor has a molecular shape similar to the substrate, so it binds at the active site and prevents the substrate from using it. Fewer enzyme–substrate complexes form, and the rate falls.
Inhibitor and substrate compete for the same site. Raising the substrate concentration means substrate molecules outnumber inhibitor molecules, so the active sites are mostly occupied by substrate and the rate recovers towards the uninhibited Vmax. Most competitive inhibitors are reversible.
Non-Competitive Inhibitors¶
A non-competitive inhibitor binds at a different place on the enzyme, the allosteric site. Binding changes the tertiary structure, so the active site changes shape and the substrate can no longer bind.
The inhibitor does not occupy the active site, so more substrate cannot displace it. A fixed fraction of the enzyme molecules is out of action whatever the substrate concentration, and Vmax is lowered.
Exam technique
Use the vocabulary exactly. A competitive inhibitor competes with the substrate for the active site, and "it blocks the enzyme" is too vague to show that. For a graph question, state what happens to Vmax: unchanged but reached at a higher substrate concentration for a competitive inhibitor, and lowered for a non-competitive one.
Reversible vs Irreversible Inhibition¶
The difference matters in medicine and in toxicology. Many drugs and poisons are irreversible inhibitors. Organophosphate nerve agents, for example, form a permanent covalent bond with acetylcholinesterase, so the neurotransmitter acetylcholine is no longer broken down.
End-Product Inhibition¶
In a metabolic pathway, the final product can inhibit an enzyme early in the pathway, often by binding at an allosteric site. When enough product has built up, it switches off its own production. When the product level falls, the inhibitor leaves and the pathway restarts. This is negative feedback, and it stops the cell from wasting resources on a molecule it already has enough of.
Interpreting Enzyme Data¶
Enzyme activity is usually measured indirectly, so the first step is to identify what the measured variable stands for. A colour change can track the substrate disappearing, as when the blue-black colour of iodine and starch fades while amylase breaks the starch down. It can also track the product appearing, as when a gas from the reaction is collected. The rate is the amount of product formed, or substrate used, per unit time.
| Graph | Shape | Reason |
|---|---|---|
| Rate against temperature | Rises to an optimum, then falls steeply | More collisions up to the optimum, then denaturation |
| Rate against pH | Bell-shaped, peaking at the optimum | Ionic and hydrogen bonds disrupted on either side of it |
| Rate against substrate concentration | Rises, then plateaus at Vmax | All active sites occupied. With a non-competitive inhibitor the plateau is lower. With a competitive inhibitor the same plateau is reached, but at a higher concentration |
| Rate against enzyme concentration, substrate in excess | Straight line through the origin, then a plateau | More active sites, until substrate becomes limiting |
Worked example: calculating a rate
Catalase breaks down hydrogen peroxide and releases oxygen gas. A student collects 18 cm³ of oxygen in the first 30 s.
Rate = volume ÷ time = 18 cm³ ÷ 30 s = 0.60 cm³ s⁻¹
Use the initial rate, from the start of the reaction. The substrate is used up as the reaction goes on, so the rate falls and a later average would be too low. If the curve is not straight at the start, draw a tangent at time zero and use its gradient.
Worked example: identifying an inhibitor from data
A student measures the rate of a reaction at six substrate concentrations with no inhibitor, with inhibitor A and with inhibitor B.
| Substrate concentration / mmol dm⁻³ | 1 | 2 | 5 | 10 | 20 | 50 |
|---|---|---|---|---|---|---|
| Rate, no inhibitor / units min⁻¹ | 5 | 9 | 15 | 18 | 19 | 20 |
| Rate with A / units min⁻¹ | 2 | 4 | 9 | 14 | 17 | 19 |
| Rate with B / units min⁻¹ | 2.5 | 4.5 | 7.5 | 9 | 9.5 | 10 |
Inhibitor A lowers the rate at low substrate concentration, but at 50 mmol dm⁻³ the rate is 19, close to the uninhibited 20. Extra substrate has overcome the inhibitor, so A is competitive.
Inhibitor B halves the rate at every concentration and levels off at 10, half the uninhibited Vmax. Extra substrate does not restore the rate, so B is non-competitive.
Common Confusions¶
A plateau at high substrate concentration does not mean the enzyme has stopped working. Every active site is occupied at once, so enzyme concentration has become the limiting factor. The enzyme is working at its maximum rate.
Denaturation and inhibition are different. Denaturation permanently changes the tertiary structure and destroys the active site. Reversible inhibition is temporary, and the enzyme recovers when the inhibitor leaves.
Extra substrate restores the full Vmax in the presence of a competitive inhibitor, because substrate outcompetes it. It cannot with a non-competitive inhibitor, because the inhibited enzyme molecules are out of action whatever the substrate concentration, so Vmax stays lower.
Cold does not denature enzymes. At low temperatures enzymes are slow because collisions are rare and gentle, but their structure is intact and activity returns as they warm. Above the optimum the rate falls because denaturation outweighs the benefit of extra collisions, and cooling does not reverse it.
Check Yourself¶
- Define activation energy and state how an enzyme affects it.
- The rate of an enzyme-catalysed reaction is 4.0 units min⁻¹ at 20 °C and 7.2 units min⁻¹ at 30 °C. Calculate Q10 and state what it shows.
- Explain why the rate of an enzyme-catalysed reaction falls at temperatures above the optimum.
- A student says: "The rate stops rising at high substrate concentration because the enzyme has been denatured." Explain why this is wrong and what is actually happening.
- A drug is added to an enzyme-catalysed reaction. At low substrate concentration the rate falls, but at very high substrate concentration the rate matches that of the reaction without the drug. Identify the type of inhibition and explain the result.
- A cell makes an amino acid in a four-step pathway. Explain how end-product inhibition stops the cell making more than it needs.
Answers
- Activation energy is the minimum energy needed to start a reaction by breaking the existing bonds. An enzyme lowers it, by binding the substrate in its active site, which strains the bonds or holds the reactants in the right orientation.
- Q10 = R2 ÷ R1 = 7.2 ÷ 4.0 = 1.8. The rate rises by a factor of 1.8 for each 10 °C rise in temperature.
- Above the optimum, the extra kinetic energy breaks the bonds that hold the tertiary structure in shape. The active site changes shape, so the substrate can no longer bind and fewer enzyme–substrate complexes form. Denaturation outweighs the increase in collisions, so the rate falls.
- The enzyme is not denatured. At high substrate concentration every active site is occupied, so the enzyme is saturated and working at Vmax. Enzyme concentration is now the limiting factor, so extra substrate has no further effect.
- It is competitive inhibition. The inhibitor competes with the substrate for the active site. At high substrate concentration, substrate molecules far outnumber the inhibitor, so the active sites are mostly occupied by substrate and Vmax is reached as it is without the inhibitor.
- The amino acid, the end product, binds to an enzyme early in the pathway, often at an allosteric site. This changes the active site, so the enzyme works more slowly and less product is made. When the amino acid level falls, the inhibitor leaves and the pathway restarts. This is negative feedback.
Key Terms¶
- Enzyme: a globular protein that acts as a biological catalyst, speeding up chemical reactions by lowering their activation energy.
- Active site: the region of an enzyme where a complementary substrate binds.
- Substrate: the molecule upon which an enzyme acts.
- Activation energy: the minimum energy required for a chemical reaction to proceed.
- Enzyme–substrate complex: the temporary structure formed when a substrate binds to the active site of an enzyme.
- Lock-and-key model: a model of enzyme action in which a specific substrate fits rigidly into a pre-formed, complementary active site.
- Induced-fit model: a model of enzyme action in which substrate binding causes the active site to change shape slightly so it becomes more complementary, helping catalysis.
- Intracellular enzyme: an enzyme that acts within the cell that produced it.
- Extracellular enzyme: an enzyme that is secreted and acts outside its producing cell.
- Denaturation: an irreversible change to a protein's tertiary structure, altering the active site so the substrate can no longer bind.
- Optimum: the temperature or pH at which an enzyme's activity is greatest.
- Q10: the factor by which reaction rate changes for a 10 °C temperature rise.
- Limiting factor: the factor in shortest supply that restricts the rate of a process.
- Saturation point: the substrate concentration at which all enzyme active sites are occupied and rate plateaus.
- Cofactor: a non-protein molecule or ion required for an enzyme to be active.
- Coenzyme: an organic cofactor that temporarily associates with an enzyme.
- Prosthetic group: a cofactor permanently bound to an enzyme.
- Competitive inhibitor: a molecule that competes with the substrate for the active site.
- Non-competitive inhibitor: a molecule that binds away from the active site and changes active-site shape.
- Allosteric site: a region of an enzyme away from the active site where regulatory molecules bind.
- End-product inhibition: regulation in which the final product of a metabolic pathway inhibits an early enzyme in that pathway, acting as a negative feedback mechanism.
- Reversible inhibition: inhibition in which the inhibitor forms weak bonds and can dissociate from the enzyme.
- Irreversible inhibition: inhibition in which the inhibitor forms permanent covalent bonds, permanently disabling the enzyme.