Enzymes and metabolism

📚The biology

Enzymes as catalysts

An enzyme is a biological catalyst: it increases the rate of a reaction without being used up or permanently changed. Most reactions in cells would happen far too slowly at body temperature to sustain life. Enzymes speed them up enormously — often by factors of millions — at the moderate temperatures and pH found in cells, where the alternative of heating would destroy the cell.

Enzymes and metabolism

Metabolism is the complex network of interdependent and interacting chemical reactions occurring in a living organism. Each enzyme is specific, catalysing one reaction or one type of reaction, so a cell needs thousands of different enzymes. That specificity is also what allows control: by making more or less of an enzyme, or by activating or inhibiting it, a cell can speed up or slow down one particular reaction without affecting the others.

Anabolic and catabolic reactions

Enzymes and the active site

Enzymes are globular proteins. The part of the enzyme where the substrate binds and the reaction is catalysed is the active site. Only a few amino acids make up the active site itself, but the rest of the protein matters too: interactions between amino acids throughout the three-dimensional structure hold the active site in exactly the right shape and give it the right chemical properties for catalysis (B1.2).

Induced fit

The substrate binds to the active site because their shapes and chemical properties are complementary. Binding is not like a rigid key in a lock. When the substrate enters the active site, both the enzyme and the substrate change shape slightly, so that they fit more closely. This is the induced-fit model. The change in shape puts strain on bonds within the substrate, making them easier to break, or brings two substrates into the right position to react. The products are then released and the enzyme returns to its original shape, ready for another substrate.

Movement and collisions

A substrate and an active site must come together before a reaction can happen. In solution, substrate and enzyme molecules are in constant random motion and collide by chance. Each collision in which the substrate meets the active site with the correct orientation can lead to binding.

Sometimes one of the partners does not move much. Large substrate molecules, such as starch or cellulose, may be effectively immobilized, so the enzymes do the moving. Some enzymes are immobilized by being embedded in membranes, such as the enzymes of the electron transport chain or ATP synthase, so the substrates move to them.

Active site, specificity and denaturation

These three ideas are linked through shape:

• Specificity exists because only a substrate with a shape and chemistry complementary to the active site can bind.
• Denaturation is a change in the three-dimensional structure of the protein, so the shape of the active site changes. The substrate no longer fits, and the enzyme can no longer catalyse the reaction. Denaturation is usually permanent.

Temperature, pH and substrate concentration

Each effect is explained using collision theory and denaturation.

Temperature. As temperature rises, enzyme and substrate molecules move faster, so collisions between substrates and active sites happen more often and with more energy, and the rate increases. Above the optimum, the enzyme vibrates so much that bonds holding its tertiary structure break; the active site changes shape and the enzyme is denatured. The rate falls steeply, often to zero. The graph rises gradually to a peak and then drops sharply.

pH. Each enzyme has an optimum pH, at which the charges on the R-groups in and around the active site are right for binding and catalysis. On either side of the optimum, H⁺ ions alter those charges, breaking ionic and hydrogen bonds, so the active site changes shape; at extreme pH the enzyme is denatured. The graph is roughly a symmetrical peak. Most intracellular enzymes have an optimum near pH 7; pepsin, in the stomach, works best near pH 2.

Substrate concentration. At low substrate concentrations, raising the concentration increases the frequency of collisions between substrates and active sites, so the rate rises steeply. As concentration increases further, more and more active sites are occupied at any moment, so extra substrate has less effect. Eventually all active sites are occupied — the enzyme is saturated — and the rate reaches a maximum and levels off. Enzyme concentration is now the limiting factor.

Sketch graphs like these are models: generalized shapes describing the relationships. They can be tested and evaluated against real experimental results, which will never follow the idealized curve exactly.

Measuring the rate of an enzyme-catalysed reaction

Rate is the change in amount of substrate or product per unit time. It can be measured by following:

• the production of a product — for example the volume of oxygen released when catalase breaks down hydrogen peroxide, collected in a gas syringe or measured by displacing water;
• the disappearance of a substrate — for example starch broken down by amylase, tested with iodine at intervals, or followed by the decreasing colour intensity using a colorimeter.

From a table or graph of product against time, the rate over an interval is the change divided by the time taken. The initial rate, before substrate starts to run out, is the steepest part of the curve and is the best measure for comparing conditions. Where the time taken to reach an end point is measured instead, rate = 1 / time.

Activation energy

Every reaction needs an input of energy to start: energy is required to break bonds within the substrate. This is the activation energy. As new bonds form in the products, energy is released. In an exergonic reaction, more energy is released than was put in.

Enzymes work by lowering the activation energy. They do not change the energy of the substrates or of the products, or the overall energy change of the reaction. On an energy diagram (energy against progress of reaction), the curve with an enzyme has a lower peak, but starts and ends at the same levels as the curve without. With a lower activation energy, a much larger proportion of collisions have enough energy to react, so the rate increases.

Intracellular and extracellular enzymes AHL

Heat from metabolism AHL

No metabolic reaction is 100% efficient at transferring energy: some energy is always converted to heat. Heat generation is therefore an inevitable consequence of metabolism. Mammals and birds, and some other animals, depend on this heat to maintain a constant, high body temperature (D3.3).

Linear and cyclical pathways AHL

Metabolic reactions are organized into pathways, in which the product of one enzyme-catalysed step is the substrate of the next.

Non-competitive inhibition AHL

Some enzymes have an allosteric site, a binding site separate from the active site. Only specific substances can bind to it. When an inhibitor binds to the allosteric site, it causes interactions within the enzyme that change its conformation, altering the active site enough to prevent catalysis. Binding is reversible.

Because the inhibitor does not compete for the active site, adding more substrate does not overcome the inhibition: the inhibited enzyme molecules are out of action whatever the substrate concentration. The maximum rate is reduced.

Competitive inhibition AHL

A competitive inhibitor has a shape similar to the substrate and binds reversibly to the active site, blocking it. Substrate and inhibitor compete for the same site; which one binds depends on their relative concentrations.

At high substrate concentration, substrate molecules win most of the collisions, so increasing substrate concentration reduces the effect of the inhibitor, and the same maximum rate can eventually be reached.

Statins, drugs used to lower blood cholesterol, are competitive inhibitors of a liver enzyme (HMG-CoA reductase) in the pathway that synthesizes cholesterol. They resemble the enzyme’s substrate, bind to its active site, and reduce the rate of cholesterol production.

Feedback inhibition AHL

In feedback inhibition, the end product of a metabolic pathway inhibits an enzyme near the start of the pathway, usually by binding to an allosteric site.

The pathway that makes the amino acid isoleucine from threonine is the standard example. When isoleucine accumulates, it binds to the allosteric site of threonine deaminase, the first enzyme of the pathway, inhibiting it. The whole pathway slows down, so less isoleucine is made. When isoleucine is used up and its concentration falls, it detaches, and the pathway speeds up again. The cell makes exactly as much isoleucine as it needs, without wasting energy and raw materials on intermediates.

Mechanism-based inhibition AHL

In mechanism-based inhibition, the inhibitor binds to the active site and a chemical change occurs that attaches it irreversibly, permanently inactivating the enzyme.

Penicillin is an example. Bacteria use enzymes called transpeptidases to form cross-links in their cell walls. Penicillin binds to the active site of transpeptidase and becomes covalently bonded to it, so the enzyme can no longer build cross-links. The bacterial wall is weakened, and the cell bursts as water enters by osmosis.

Some bacteria are resistant to penicillin because they have a modified transpeptidase whose active site no longer binds penicillin, while still carrying out its normal function (C3.2).