Enzyme action

Enzymes speed up chemical reactions in living organisms. They are biological catalysts: they increase reaction rates without being consumed, so they can be used again. Most enzymes are proteins.

A reaction requires an initial input of energy before it can proceed. This is its activation energy. An enzyme lowers the activation energy of the reaction it catalyses, allowing the reaction to occur more rapidly.

Enzymes catalyse reactions both inside cells (intracellular) and outside cells (extracellular). These reactions contribute to the structures and functions of living organisms, from individual cells to the whole organism.

Enzyme action at the active site

A protein enzyme folds into a particular three-dimensional form, called its tertiary structure. This structure determines the shape and chemical properties of a region called the active site.

A substrate is a molecule that an enzyme acts on. A substrate with a complementary shape and chemical properties can bind to the active site, forming an enzyme–substrate complex. This selectivity gives an enzyme specificity for particular substrates and the reactions it catalyses. After the reaction, the products leave the active site and the enzyme can act again.

Models of enzyme–substrate binding

The earlier lock-and-key model represented the active site as a rigid shape that was already exactly complementary to the substrate. The substrate fitted into it without the active site changing shape.

The induced-fit model develops this explanation: when a substrate binds, the active site changes shape, improving the fit and helping the enzyme catalyse the reaction. The active site is therefore not treated as a rigid structure.

Effect of enzyme concentration on reaction rate

Increasing the concentration of an enzyme means there are more enzyme molecules in a given volume. Each enzyme molecule has an active site where a substrate can bind. More enzyme molecules therefore provide more active sites for substrate molecules to bind to at the same time.

When a substrate binds to an active site, it forms an enzyme–substrate complex. If enough substrate is available, adding enzyme allows more complexes to form per unit time. More substrate is converted into product per unit time, so the reaction rate increases, provided other conditions remain constant.

When substrate becomes limiting

If enzyme concentration continues to increase while the amount of substrate remains limited, there may not be enough substrate molecules to occupy all the available active sites. Substrate availability is then limiting: adding more enzyme has less effect on the reaction rate because the extra active sites cannot all be supplied with substrate.

Effect of substrate concentration on reaction rate

At a fixed enzyme concentration, increasing the substrate concentration initially increases the reaction rate. More substrate molecules are available to bind to the enzymes, so enzyme–substrate complexes form more frequently and more substrate is converted to product per unit time.

Each enzyme has an active site where its substrate binds. As substrate concentration rises, a greater proportion of the available active sites are occupied. The rate therefore continues to increase, but by a smaller amount as fewer active sites remain available.

Eventually, the enzymes become saturated: their active sites are occupied as soon as they become available. The reaction has reached its maximum rate for that enzyme concentration. Adding more substrate cannot increase the rate further, because the enzymes are already working at their maximum rate. More enzyme would need to become available to increase the maximum rate.

Competitive and non-competitive enzyme inhibition

An enzyme inhibitor reduces the rate of an enzyme-controlled reaction. Competitive and non-competitive inhibitors do this in different ways: they bind at different sites on the enzyme and respond differently to an increase in substrate concentration.

Competitive inhibition

A substrate normally binds to an enzyme’s active site, where the reaction is catalysed. A competitive inhibitor binds to that same site, so the substrate and inhibitor compete to occupy it. While the inhibitor occupies the active site, the substrate cannot bind there.

Increasing the competitive inhibitor concentration increases competition for active sites and reduces the reaction rate. Increasing the substrate concentration can reduce the inhibitor’s effect: substrate molecules are then more likely to occupy active sites before inhibitor molecules bind.

Non-competitive inhibition

A non-competitive inhibitor binds to the enzyme at a site other than the active site. This alters the enzyme so that it catalyses the reaction less effectively. Increasing the concentration of a non-competitive inhibitor generally increases inhibition and further reduces the reaction rate.

Increasing substrate concentration does not overcome non-competitive inhibition. The inhibitor does not compete with the substrate for the active site, so having more substrate available does not prevent the inhibitor from binding at its separate site.

Effect of pH on enzyme activity

An enzyme works fastest at a particular pH under the conditions considered. This is its optimum pH. Moving above or below the optimum generally reduces the rate of the reaction.

Changes in pH alter the electrical charges on groups within the enzyme. This can disrupt interactions that help maintain the enzyme’s tertiary structure, its overall three-dimensional shape. It can also change the shape or chemical properties of the active site, where the substrate binds and the reaction is catalysed.

If the active site changes, the substrate may bind less effectively, or the enzyme may be less able to catalyse the reaction after the substrate binds. In either case, the reaction rate falls.

At extreme pH, the structural changes can be large enough to denature the enzyme. Denaturation causes a lasting change in its structure, including the active site, so the enzyme loses its activity.

Effect of temperature on enzyme activity

Temperature affects how often enzyme and substrate molecules collide and whether the enzyme keeps the shape needed to bind its substrate. As temperature rises, the reaction rate usually increases to a maximum, then falls at higher temperatures.

Temperature increase up to the optimum

At low temperatures, enzyme and substrate molecules have relatively little kinetic energy and move slowly. Successful collisions are less frequent, so fewer enzyme–substrate complexes form each second and the reaction is slow. Low temperatures do not normally denature the enzyme, so its activity can increase again when it is warmed.

As temperature rises, the molecules gain kinetic energy and move faster. Successful collisions become more frequent, so more enzyme–substrate complexes form each second and the reaction rate usually increases. The temperature at which the reaction rate is highest is the enzyme’s optimum temperature.

Temperature above the optimum

At higher temperatures, interactions that maintain the enzyme’s three-dimensional tertiary structure are increasingly disrupted. This changes the shape of the enzyme, including its active site. The enzyme is then described as denatured.

The changed active site is no longer complementary to the substrate, so fewer enzyme–substrate complexes form. Although molecules are moving faster, the increasing loss of functional active sites causes the reaction rate to fall above the optimum.

Investigating enzyme-controlled reaction rates

An enzyme-controlled reaction can be investigated by changing one condition and measuring how quickly the reaction takes place. The condition deliberately changed is the independent variable; the reaction rate is the dependent variable. Other relevant conditions are kept the same so that differences in rate can be linked to the independent variable.

For example, an investigation could test how pH affects catalase activity. Catalase breaks down hydrogen peroxide and produces oxygen. Use solutions at different pH values and measure the volume of oxygen produced over the same time at each pH. Keep conditions such as temperature, enzyme concentration and starting hydrogen peroxide concentration the same, and use the same method and apparatus to measure oxygen each time.

Measuring reaction rate

Reaction rate describes how much substrate is used up, or how much product is formed, per unit of time. For a product measurement:

A rate can also be calculated by dividing the amount of substrate used up by the time taken. In the catalase investigation, collecting $$12\ \text{cm}^3$$ of oxygen in $$2\ \text{min}$$ gives an average rate of $$6\ \text{cm}^3/\text{min}$$ over that period. Use the same measurement method and time period for each pH so the rates can be compared.

Repeats and interpretation

Repeat the investigation at each value of the independent variable. Repeats show how much measurements vary and help identify an anomalous result: one that differs markedly from the others under the same conditions. Calculate a mean rate for each value to represent the repeats; investigate an anomaly before deciding whether there is a reason to exclude it.

Plot the independent variable on the horizontal axis and reaction rate on the vertical axis. The pattern shows how enzyme activity changes as the independent variable changes. For example, the pH with the highest measured rate is the pH at which catalase was most active among those tested.