Enzymes

What is metabolism?

Every living cell is busy. Inside a single cell, thousands of chemical reactions are happening at the same time. Some reactions break large molecules down to release energy. Others build large molecules, such as proteins and DNA, from smaller ones. All of the chemical reactions that happen in a cell or an organism are called metabolism.

Most of these reactions do not happen on their own. They are linked together in a sequence called a metabolic pathway. The product of the first reaction becomes the starting molecule for the second reaction, the product of the second reaction becomes the starting molecule for the third, and so on.

The important point for this topic is that every single step in a metabolic pathway is controlled by its own enzyme. That is exactly what the specification means when it describes metabolism as a series of enzyme controlled reactions.

A simple way to picture this is a sandwich shop production line. One person slices the bread, the next adds the filling and the last one wraps it. If the person adding the filling is off sick, sliced bread piles up and no finished sandwiches come out. A metabolic pathway behaves in the same way. If enzyme 2 is missing or not working, molecule B builds up and molecules C and D are not made.

You will meet many enzyme controlled pathways later in the course, including respiration and photosynthesis in Unit 3 and DNA replication in the nucleic acids topic. For now, the idea to hold on to is simple: no enzymes, no metabolism.

What are enzymes made of?

Enzymes are proteins. To understand how enzymes work, you first need a quick reminder of how proteins are built, because the shape of the protein is what makes an enzyme work.

A protein is made from amino acids joined together by peptide bonds in condensation reactions. There are 20 different amino acids. They differ only in their R group, which is the variable part of the molecule. Some R groups carry a positive or negative charge, some are polar and some are non-polar.

Enzymes are globular proteins. This means the chain is folded into a compact, roughly spherical shape. Most of the hydrophilic (water loving) R groups face outwards, so enzymes are soluble. This lets them dissolve in the cytoplasm of a cell or in digestive juices, where they can meet their substrate.

Here is the chain of cause and effect that the rest of this topic depends on. The sequence of amino acids decides where the bonds form. Where the bonds form decides the tertiary structure. The tertiary structure decides the exact shape of the active site, which is the part of the enzyme where the substrate binds. So if one important amino acid is swapped for a different one, the active site can end up the wrong shape and the enzyme may stop working.

Do enzymes work inside or outside cells?

All enzymes are made inside cells, on ribosomes, during protein synthesis. What differs is where they go on to do their job. Biologists sort enzymes into two groups depending on where they act.

Intracellular enzymes act inside the cell that made them. Many are dissolved in the cytoplasm, some are inside organelles and some are attached to membranes. For example, catalase breaks down hydrogen peroxide, a toxic by-product of metabolism, into water and oxygen before it can damage the cell. DNA polymerase works inside the nucleus, and the enzymes of respiration work in the cytoplasm and mitochondria.

Extracellular enzymes are made inside the cell but are then secreted out of it by exocytosis, so they act outside the cell. Salivary amylase is made by cells in the salivary glands but works in the mouth. Trypsin is made in the pancreas but works in the small intestine. Lysozyme is secreted into tears and saliva.

A simple example of why extracellular enzymes are useful is mould growing on a slice of bread. The fungus cannot swallow the bread. Instead, its hyphae secrete enzymes onto the bread. The enzymes break the large starch and protein molecules into small soluble molecules, which the fungus then absorbs. You will meet this again as saprotrophic nutrition in Unit 2.

What is an active site and why are enzymes specific?

When an enzyme folds into its tertiary structure, a small region of its surface forms a pocket or groove. This region is called the active site. It is made from only a few of the enzyme's amino acids, but their R groups are positioned very precisely so that they can bind to the substrate.

The shape of the active site is complementary to the shape of the substrate. Complementary means the two shapes fit together, a bit like two neighbouring jigsaw pieces. The charges on the R groups in the active site also match up with charges on the substrate, which helps it bind.

Enzyme and substrate molecules move around randomly. A reaction can only happen when a substrate molecule collides with the active site the right way round. When this happens it is called a successful collision, and the substrate binds to form an enzyme-substrate complex.

Because each enzyme has an active site with a particular shape, only one type of substrate, or a small group of very similar substrates, will fit. We say enzymes are specific. For example, the enzyme tyrosinase converts the amino acid tyrosine into the pigment melanin. Other amino acids do not have a shape that is complementary to its active site, so tyrosinase cannot act on them.

The simplest model of how this works is the lock and key model. The substrate is like a key and the active site is like a lock. Only the correctly shaped key fits the lock. This model is useful for explaining specificity, but it treats the active site as completely rigid. The next section shows why scientists now think the active site is slightly flexible.

What is the induced fit theory?

The lock and key model suggests the active site is a fixed, rigid shape. Evidence from studying enzyme structures shows that this is not quite true. The active site can change shape slightly. The induced fit theory explains enzyme action using this idea.

In the induced fit theory, the active site is not an exact fit for the substrate to begin with. As the substrate enters the active site, the active site changes shape slightly and moulds itself tightly around the substrate. The substrate has induced, or caused, the change in shape. This tighter fit puts strain on particular bonds in the substrate, which makes them easier to break. This is one way an enzyme lowers the activation energy of the reaction.

A good everyday example is a woollen glove. When it is lying on a table it is roughly hand shaped but floppy. When you push your hand in, the glove stretches and fits closely around your fingers. The active site behaves in a similar way when the substrate enters.

The specification uses lysozyme to illustrate induced fit. Lysozyme is an enzyme found in tears, saliva and mucus. It helps to defend the body against bacteria. It works by hydrolysing glycosidic bonds in the long polysaccharide chains that make up bacterial cell walls. Hydrolysis means breaking a bond by adding water.

The active site of lysozyme is a long groove. When part of a polysaccharide chain from the cell wall fits into the groove, the enzyme changes shape and closes slightly around it. This distorts one of the sugar units in the chain and puts strain on a glycosidic bond. The strained bond is then hydrolysed, splitting the chain.

The final step links to osmosis from the membranes topic. The cytoplasm of the bacterium has a lower (more negative) water potential than the tears or saliva around it. Normally the strong cell wall stops the cell from bursting as water moves in. Once lysozyme has weakened the wall, water moves into the cell by osmosis and the cell bursts.

How do enzymes speed up reactions?

Catalysis means speeding up a chemical reaction using a catalyst. The catalyst is not used up and is not permanently changed by the reaction, so it can be used again. Enzymes are biological catalysts.

To understand how enzymes speed up reactions, you need the idea of activation energy. Activation energy is the minimum amount of energy that must be supplied to start a reaction, so that bonds in the substrate can break.

Think of pushing a heavy trolley over a small hump before it can roll down a slope on the other side. Even though the trolley ends up lower down, you still have to push it up and over the hump first. Activation energy is like that hump.

Many reactions in cells have a high activation energy. At body temperature, very few molecules have enough energy to get over this barrier, so the reaction would be far too slow to keep the organism alive. Heating the cell to give molecules more energy is not an option, because high temperatures would denature its proteins.

Enzymes solve this problem by providing a different route for the reaction that has a lower activation energy. With a lower barrier to get over, many more substrate molecules can react at body temperature, so the reaction happens much faster.

The graph below is the standard way of showing this. Read through the steps underneath it carefully, because WJEC has asked candidates both to label this kind of graph and to draw the enzyme curve onto it.

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How does binding in the active site lower the activation energy? When substrates are held in the active site, they are brought close together in the right position to react. In the induced fit theory, the active site also moulds around the substrate and puts strain on bonds, which makes them easier to break. Both of these effects mean less energy is needed to start the reaction.

Catalase is a good example of why this matters. Hydrogen peroxide is made during respiration and is toxic. It does break down on its own, but very slowly. Catalase lowers the activation energy so that hydrogen peroxide is broken down into water and oxygen fast enough at body temperature to stop it building up and damaging cells.

How do we measure the rate of an enzyme reaction?

The rate of a reaction tells you how quickly it is happening. For an enzyme controlled reaction, rate is the amount of product made, or the amount of substrate used up, in a given amount of time. Before you can compare the effect of temperature, pH or concentration, you need to know how rate is measured.

When you measure product over time, you usually get a curve that is steep at the start and then levels off. The graph below shows an example: the volume of oxygen released when catalase breaks down hydrogen peroxide.

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A tangent is a straight line that just touches the curve at one point and has the same steepness as the curve at that point. To find the rate at a particular time, draw a tangent at that time, choose two points on the tangent that are far apart, and divide the change in the y value by the change in the x value.

How does temperature affect enzyme activity?

Temperature affects enzyme activity in two different ways. Up to a certain point it speeds reactions up. Beyond that point it damages the enzyme. The easiest way to understand this is to go through it in stages from cold to hot.

Stage 1, below the optimum. As temperature rises, enzyme and substrate molecules gain kinetic energy and move around faster. They collide more often, so there are more successful collisions and more enzyme-substrate complexes form each second. The rate of reaction increases.

Stage 2, the optimum. The optimum temperature is the temperature at which the rate of reaction is highest. For many enzymes in the human body the optimum is close to body temperature, about 37 °C. Enzymes from other organisms can be very different. Some bacteria that live in hot springs have enzymes with an optimum above 70 °C.

Stage 3, above the optimum. The molecules now have so much kinetic energy that the enzyme vibrates strongly. This breaks the hydrogen bonds and ionic bonds that hold its tertiary structure in place. The tertiary structure changes, so the shape of the active site changes. The active site is no longer complementary to the substrate, so fewer enzyme-substrate complexes can form and the rate falls quickly. The enzyme has been denatured, and this change is permanent.

What about low temperatures? At low temperatures enzymes are inactive rather than denatured. The molecules have very little kinetic energy, so there are few collisions and few enzyme-substrate complexes, but the shape of the active site is unchanged. If the temperature rises again, the enzyme works again. This is why freezing food slows down decay without destroying the enzymes.

The graph below shows the typical shape. Work through the notes under it until you can explain every part of the curve.

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A memorable example comes from WJEC's 2025 paper. In Siamese cats, the enzyme tyrosinase makes the dark pigment melanin, but it is denatured above 35 °C. The cat's ears, face, paws and tail are cooler than 35 °C, so tyrosinase works there and the fur is dark. The main part of the body is warmer than 35 °C, so tyrosinase is denatured, little melanin is made and the fur is pale.

How does pH affect enzymes, and what does a buffer do?

pH is a measure of how acidic or alkaline a solution is. It depends on the concentration of hydrogen ions (H⁺). A low pH, such as pH 2, means a high concentration of hydrogen ions, so the solution is acidic. A high pH, such as pH 10, means a low concentration of hydrogen ions, so the solution is alkaline. pH 7 is neutral.

Remember that some amino acid R groups carry electrical charges, and that ionic bonds and hydrogen bonds between R groups help hold the tertiary structure together. Hydrogen ions and hydroxide ions interact with these charged groups. When the pH changes, the charges on the R groups change.

This has two effects. First, the charges on the R groups inside the active site change, so the substrate may no longer be attracted to and held in the active site. Second, if the pH moves far from the optimum, the ionic and hydrogen bonds holding the tertiary structure break, the active site changes shape and the enzyme is denatured. Either way, fewer enzyme-substrate complexes form and the rate falls.

Each enzyme has an optimum pH at which its rate is highest. This usually matches the place where the enzyme works. Pepsin works in the stomach, which is very acidic, and has an optimum of about pH 2. Salivary amylase works in the mouth and has an optimum of about pH 7. Trypsin works in the small intestine and has an optimum of about pH 8.

The graph below compares two enzymes. Notice that each one has its own curve, and read the notes underneath.

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A buffer is a solution that resists changes in pH when small amounts of acid or alkali are added. In other words, it keeps the pH constant. Buffers matter in two places. In the body, fluids such as blood are buffered so that enzymes keep working. In the laboratory, a buffer at a chosen pH is added to an enzyme experiment so that pH stays the same in every test and cannot affect the results.

Why would the pH change during an experiment anyway? Some reactions produce acidic products. For example, when lipase breaks down fats, fatty acids are made and they lower the pH. Without a buffer, the pH would drift during the experiment and change the rate for a reason that has nothing to do with the variable being tested.

How does substrate concentration affect rate?

Imagine a fixed amount of enzyme, so there is a fixed number of active sites. Now think about what happens as you add more and more substrate.

At low substrate concentrations, many active sites are empty at any moment. Adding more substrate molecules means more collisions with active sites, so more enzyme-substrate complexes form each second and the rate increases. Here, the substrate concentration is the limiting factor. A limiting factor is the factor that is in shortest supply and so holds back the rate.

At high substrate concentrations, almost every active site is occupied at any moment. We say the active sites are saturated. Adding even more substrate cannot make the reaction any faster, because there are no free active sites for the extra substrate to bind to. The rate stays constant at its maximum. Now the enzyme concentration, meaning the number of active sites, is the limiting factor.

A car wash with four washing bays is a helpful example. If only one car turns up every ten minutes, the number of cars washed per hour depends on how many cars arrive. Once cars are arriving faster than the bays can handle, all four bays are busy all the time. Extra cars simply queue, and the number of cars washed per hour stops rising. The bays are the active sites and the cars are the substrate.

The graph below shows this relationship. It is one of the most frequently examined graphs in this topic.

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How does enzyme concentration affect rate?

Now do the opposite. Keep the substrate concentration high and increase the amount of enzyme. If there is plenty of substrate, which we call substrate in excess, every extra enzyme molecule provides extra active sites that will quickly be filled. More active sites means more enzyme-substrate complexes per second, so the rate increases in direct proportion to the enzyme concentration. Doubling the enzyme doubles the rate, and the graph is a straight line.

If the amount of substrate is limited, something different happens at high enzyme concentrations. There are now more active sites than there are substrate molecules to fill them. Adding more enzyme just adds more empty active sites, so the rate levels off. The substrate concentration has become the limiting factor.

The graph below shows both situations on one set of axes.

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What are competitive and non-competitive inhibitors?

An enzyme inhibitor is a molecule that reduces the rate of an enzyme controlled reaction. It does this by reducing the number of enzyme-substrate complexes that can form. The specification asks you to understand the principles of two types: competitive and non-competitive inhibition.

A competitive inhibitor has a shape similar to the substrate. This means it is also complementary to the active site, so it can bind to the active site. While it is there, the substrate cannot bind. The inhibitor and the substrate are competing for the same active sites, which is where the name comes from.

Because it is a competition, the effect depends on how much of each molecule is present. If you add more substrate, a substrate molecule is more likely than an inhibitor molecule to collide with any free active site. At very high substrate concentrations the inhibitor has very little effect, and the rate gets close to the same maximum rate as the reaction without an inhibitor.

A real medical example appeared in a WJEC paper. Ethylene glycol, found in antifreeze, is poisonous because the enzyme alcohol dehydrogenase converts it into a toxic product. Doctors treat the poisoning by giving the patient ethanol. Ethanol has a similar shape to ethylene glycol and competes for the active site of alcohol dehydrogenase, so less of the toxic product is made.

A non-competitive inhibitor does not bind to the active site. It binds to a different site on the enzyme, sometimes called an allosteric site. When it binds, it changes the tertiary structure of the enzyme, which changes the shape of the active site. The active site is no longer complementary to the substrate, so enzyme-substrate complexes cannot form.

Adding more substrate does not help, because the inhibitor is not competing for the active site. Every enzyme molecule with an inhibitor attached is out of action no matter how much substrate is present. So the maximum rate is lower. Copper ions from copper sulfate act as a non-competitive inhibitor of catalase, which WJEC has used in both written and practical papers.

The graph below shows how each type of inhibitor affects the substrate concentration graph you met earlier.

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What are immobilised enzymes and why does industry use them?

Scientists' understanding of enzyme structure has allowed enzymes to be used widely in industry. For example, lactase is used to make lactose-free milk for people who are lactose intolerant, and glucose oxidase is used in biosensors that measure the concentration of glucose.

The simplest way to use an enzyme is to mix it into a solution of its substrate. This causes problems. The enzyme ends up mixed with the product, so it has to be separated out or it contaminates the product. The enzyme is also lost at the end of each batch, so new enzyme has to be bought every time.

An immobilised enzyme is an enzyme that is fixed in place, so it cannot mix freely with the product. A common method is to trap the enzyme inside small beads of a jelly-like substance called alginate. The beads are packed into a column and the substrate solution is poured through. The substrate diffuses into the beads, the enzyme catalyses the reaction, and the products diffuse out and flow away. The enzyme stays behind in the column.

A WJEC question compared free lactase with lactase immobilised in alginate beads at different temperatures. Below 50 °C, the free lactase was more active. The free enzyme and substrate can move around and collide easily, while the substrate has to diffuse into the beads to reach the immobilised enzyme. Above 50 °C, the immobilised lactase was more active. The free lactase was being denatured, but immobilisation stabilised the enzyme and helped the active site keep its shape.

Biosensors use the same idea. A glucose biosensor contains immobilised glucose oxidase. The active site of glucose oxidase is specific to glucose, so the sensor responds only to glucose even in a sample such as urine that contains many other substances. The reaction makes hydrogen peroxide, and the electrode turns the amount of hydrogen peroxide into an electrical signal that is shown as a reading.

Specified practical work: enzyme investigations

The specification lists two enzyme investigations that you must carry out. The first investigates the effect of temperature or pH on enzyme activity. The second investigates the effect of enzyme or substrate concentration. The walkthroughs below cover every version, so you are prepared whichever one your school chose and whichever one appears in the exam.

These investigations also build the skills WJEC tests again and again: choosing and controlling variables, using a water bath and buffers, making dilutions, recording results in a table, calculating means and rates, plotting graphs with range bars and evaluating a method.

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When these pH results are plotted, the rate rises from 1.19 × 10⁻³ s⁻¹ at pH 4 to a peak of 5.00 × 10⁻³ s⁻¹ at pH 7, then falls to 1.52 × 10⁻³ s⁻¹ at pH 9. The optimum lies somewhere between pH 6 and pH 8. Because readings were only taken at whole pH units, the exact optimum cannot be identified. Repeating the experiment at 0.2 pH unit intervals between pH 6 and pH 8 would find it more precisely.

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Plotted against extract concentration, these rates lie close to a straight line through the origin. Each extra 20% of extract adds roughly 1.7 × 10⁻² s⁻¹ to the rate. This fits the idea that, with substrate in excess, doubling the number of active sites roughly doubles the number of enzyme-substrate complexes formed each second.

Enzymes summary: everything in one place

Use this section to check your understanding before trying the questions. If any row feels unfamiliar, go back to that section of the notes first.