C1.1 Enzymes and metabolism

A catalyst is a substance that speeds up a chemical reaction without being used up or permanently changed by it. Enzymes are biological catalysts: proteins made by cells that catalyse the reactions of life.

Why cells need fast reactions

Most reactions in a cell would happen far too slowly at body temperature to keep the cell alive. Sugars, fats and proteins are quite stable molecules. Without enzymes, the breakdown of glucose to release energy, or the joining of amino acids to make proteins, would take years. Enzymes make these reactions happen in fractions of a second, so that:

• energy can be released from food as fast as cells need it, for example in contracting muscle;
• molecules can be built quickly enough for growth, repair and reproduction;
• reactions can happen at the moderate temperatures of living organisms, rather than needing heat that would damage cells.

Metabolism is a network

Metabolism is the whole set of chemical reactions in an organism. These reactions are interdependent: the product of one reaction is the substrate of the next, many products feed into several pathways, and the same molecule can be made by one pathway and used by others. Changing one reaction therefore affects many others, so metabolism behaves as a linked web rather than a list of separate reactions.

Many enzymes are needed

Enzymes are specific: each one usually catalyses only one reaction, or a group of very similar reactions, because only its own substrate fits the active site. A cell therefore needs thousands of different enzymes, each coded for by a gene, one for almost every reaction in the network.

Specificity also gives the cell control. A reaction runs only if its enzyme is present and active, so cells regulate metabolism by:

• making more or less of a particular enzyme (by switching genes on or off);
• activating or inhibiting enzymes that are already present (see the AHL sections on inhibition below).

Metabolic reactions fall into two groups.

Anabolism: building up

Anabolic reactions build larger, more complex molecules from smaller ones. Many join monomers into macromolecules by condensation reactions, in which a water molecule is released as each new bond forms. They need an input of energy, usually from ATP.

• Protein synthesis: amino acids are linked by peptide bonds on ribosomes to form polypeptides.
• Glycogen formation: glucose molecules are linked by glycosidic bonds in liver and muscle cells to store energy.
• Photosynthesis: carbon dioxide is used to build sugars, using energy from light; glucose is then linked by condensation into starch.

Catabolism: breaking down

Catabolic reactions break larger molecules into smaller ones and often release energy.

• Digestion: macromolecules in food are broken into monomers by hydrolysis reactions, in which a water molecule is used to break each bond. Starch is hydrolysed to maltose and then glucose, proteins to amino acids, and triglycerides to fatty acids and glycerol.
• Cell respiration: substrates such as glucose are oxidized to carbon dioxide and water, releasing energy that is used to make ATP.

Enzymes are globular proteins: each polypeptide is folded into a compact, roughly spherical three-dimensional shape (see B1.2 Proteins). On the surface is a pocket or cleft called the active site, where the substrate binds and the reaction takes place.

A few amino acids, held by the whole protein

The active site is made of only a few amino acids, often from parts of the polypeptide that are far apart in the sequence but brought together by folding. Their R-groups give the active site its shape and its chemical properties: charge, polarity, and groups that can donate or accept protons or form temporary bonds with the substrate.

The rest of the enzyme matters too. Interactions between amino acids throughout the protein (hydrogen bonds, ionic bonds, hydrophobic interactions and disulfide bonds) hold the active-site amino acids in exactly the right positions. Change the overall fold and the active site changes as well, which is why denaturation stops catalysis.

Induced fit

The induced-fit model describes how the substrate binds:

1. Before binding, the active site is close to, but not exactly, complementary to the substrate.
2. As the substrate binds, the enzyme changes shape: the active site closes round the substrate so that the two fit tightly.
3. The substrate also changes shape. It is held in a strained position, which weakens the bonds that are to be broken (or brings reacting groups together).
4. This enzyme–substrate complex is converted to an enzyme–product complex. The products fit less well and are released, and the enzyme returns to its original shape.

An older idea, the lock-and-key model, pictured the active site as rigid and exactly complementary to the substrate. It explains specificity, but not the shape changes that are now known to happen when substrates bind. Use the Exam diagram view above to compare the two.

Movement and collisions

An enzyme can only catalyse a reaction when a substrate molecule is in its active site, and they come together only by collision. Molecules in solution are in constant random motion, so substrate molecules and enzymes collide by chance. A collision leads to binding only if the substrate arrives at the active site in a suitable orientation and with enough energy. Anything that increases the frequency of these successful collisions, such as more substrate or a higher temperature, increases the rate.

Movement does not always need both partners to move:

• Large substrate molecules may be fixed in place, so the enzyme moves to them. For example, enzymes that copy or repair DNA move along the DNA molecule, and enzymes in seeds digest starch grains that stay where they are.
• Enzymes can be immobilized in membranes while their substrates move to them. Enzymes in the plasma membrane of microvilli in the small intestine digest disaccharides that reach them, and ATP synthase is fixed in the inner membrane of the mitochondrion.

Specificity

Each enzyme catalyses one reaction (or a few very similar ones) because its active site is complementary to its substrate in shape and chemical properties. Other molecules either do not fit or lack the right charges and groups to bind, so they are not catalysed.

Denaturation

Denaturation is a change in the 3D structure of a protein. The bonds that maintain the folded shape are much weaker than the peptide bonds of the chain, so they are disrupted by:

• High temperature: the enzyme vibrates so strongly that hydrogen bonds and other interactions break.
• Extreme pH: extra H⁺ or OH⁻ ions change the charges on R-groups, breaking ionic and hydrogen bonds.

When the fold changes, the active site changes shape, so the substrate no longer binds and catalysis stops. Heat denaturation is usually permanent: cooling the enzyme does not restore the original shape. The amino acid sequence (primary structure) is not affected.

Temperature

• Rising towards the optimum: molecules move faster, so collisions between substrate and active site are more frequent and more of them have enough energy to react. The rate increases, roughly doubling for each 10 °C rise.
• The optimum: the temperature at which the rate is highest. For many human enzymes it is about 37–40 °C; enzymes of organisms from hot springs have much higher optima.
• Above the optimum: the enzyme starts to denature. More and more active sites lose their shape, so the rate falls steeply, reaching zero when all the enzyme is denatured. The curve is therefore not symmetrical.

pH

Each enzyme has an optimum pH, and the rate falls on either side of it, giving a roughly bell-shaped curve. A small change from the optimum alters the charges on R-groups in the active site, so the substrate binds less well; a larger change denatures the enzyme. Optima match where each enzyme works:

Substrate concentration

At low substrate concentrations, many active sites are empty at any moment. Adding substrate increases the frequency of collisions with active sites, so the rate rises steeply and almost in proportion. As the concentration rises further, a larger fraction of active sites are occupied, so the rate increases less and less. Eventually all active sites are in use (the enzyme is saturated) and the rate levels off at a maximum. More substrate has no effect; only more enzyme raises the maximum.

Sketch graphs are models

The smooth curves in textbooks and in this simulation are generalized sketch graphs. They are models: simplified pictures of a relationship, without real data points or units. Like any model they can be tested against experimental data. Real results show scatter between repeats, and the optimum found for temperature depends on how long the enzyme is kept warm (denaturation takes time), so real curves often differ in detail from the sketch. If data repeatedly disagree with a sketch graph, the model has to be revised.

The rate of reaction is the amount of substrate used, or product made, per unit time:

rate = change in amount of substrate or product ÷ time taken

Typical units are cm³ s⁻¹ (gas volume), g min⁻¹ (mass) or mmol dm⁻³ s⁻¹ (concentration).

Methods

• Catalase: hydrogen peroxide is broken down into water and oxygen (2 H₂O₂ → 2 H₂O + O₂). Oxygen is collected in a gas syringe or upturned measuring cylinder and its volume read at intervals.
• Amylase: samples of a starch–amylase mixture are tested with iodine at intervals. The time taken for starch to disappear (iodine no longer turns blue-black) is recorded, and rate = 1 ÷ time (units s⁻¹).
• Colour change measured with a colorimeter, or mass lost as gas escapes.

Initial rate from a graph

When product is plotted against time, the curve is steepest at the start and then levels off as the substrate is used up. The initial rate is the gradient of a tangent drawn to the curve at time zero: pick two points on the tangent and divide the change in y by the change in x. The rate at any later time is the gradient of the tangent at that time.

Reliable data

• Change one independent variable (such as enzyme concentration or temperature) and keep the others controlled: volume and concentration of substrate, temperature, pH, time.
• Do at least three repeats for each value, then calculate a mean. Look for anomalous results and judge spread (for example with the range or standard deviation).
• With secondary data, you may be given a table or graph: calculate rates from the change over a given time, or from a tangent, and always give units.

In a chemical reaction, bonds in the substrate are broken and new bonds are made to form the products.

• Breaking bonds requires energy. Before any reaction can happen, the substrate must gain enough energy to reach an unstable transition state in which its bonds are weakened. This energy is the activation energy.
• Making bonds releases energy. As the products form, energy is given out.

In an exothermic reaction, such as the hydrolysis or oxidation reactions of catabolism, more energy is released when product bonds form than is taken in to break substrate bonds, so the products have less energy than the substrate and there is an overall energy yield.

How enzymes help

Binding in the active site lowers the activation energy, for example by straining the bonds to be broken (induced fit) or holding reacting groups close together in the right orientation. Because less energy is needed, a much larger share of collisions leads to a reaction at body temperature. The enzyme does not change the energy of the substrate or products, so it does not change how much energy the reaction releases.