objectives

Enzyme inhibition

The action of enzymes can be increased or decreased by the presence of external factors, including other compounds. These compounds help to regulate and control the catalysis that enzymes perform. Inhibitors are compounds that decrease enzymatic activity, by directly binding to the enzyme, affecting the substrate's ability to bind to the active site and catalyse the reaction.

Types of inhibitors

There are two main types of inhibitors: competitive and non-competitive. Competitive inhibitors bind directly to the active site, blocking the substrate from binding. Competitive inhibitors can bind irreversibly (permanently) through a covalent bond to one of the amino acid residues in the active site, or reversibly (temporarily) through the attraction of intermolecular forces between the inhibitor and the amino acid residues in the active site. The inhibitor and substrate 'compete' to bind to the same active site, directly reducing the enzyme's activity.

Non-competitive inhibitors bind to the enzyme, but not to the active site. Instead, they bind to another site in the enzyme called the allosteric site. When the inhibitor binds to the allosteric site, it changes the shape of the enzyme, including the active site, resulting in a decrease in the ability of the substrate from being able to bind. Non-competitive inhibitors do not compete with the substrate directly and some do not alter the active site very much, meaning that the substrate can sometimes still bind, but not as efficiently. Examples of non-competitive inhibitors include heavy metal ions such as lead, mercury and silver.

Measuring enzyme activity

Enzyme assays are the laboratory studies involving enzyme kinetics. For reactions involving a single substrate, the initial rate of reaction is dependent on the concentration of both the catalyst and the substrate, reacting in a bimolecular (HL only) mechanism. As a single enzyme molecule can be reused over and over to catalyse a reaction involving many substrate molecules, it is the concentration of the substrate that has a bigger influence on the reaction rate than the concentration of the catalyst. For enzyme assays, the rate of reaction is represented by v, which stands for velocity. The rate of the reaction can be expressed in the rate expression (HL only) below:

However, the concentration of the substrate will eventually reach a level where there are not enough enzyme molecules available to catalyse the reaction and the rate will no longer increase. The concentration of the substrate is so high that it saturates all of the enzyme molecules and the reaction rate cannot increase any further. This observation is explained by Michaelis-Menten kinetics and demonstrated in the graph below. The substrate concentration at which no further change in rate is observed is referred to as the enzyme's maximum rate, Vmax.

The Michaelis constant, Km, is the substrate concentration for half of the maximum enzymatic rate, that is ½Vmax. When the Km value is small, it indicates a very strong affinity between the enzyme and the substrate, resulting in a lower concentration of substrate required to achieve Vmax. When the value is high, it indicates weak affinity between the enzyme and the substrate, requiring a higher substrate concentration to reach Vmax. The value of Km for a given enzyme-substrate complex can vary slightly with changes in temperature or pH.

When an inhibitor is present, the activity of the enzyme changes, which can be used to determine if the inhibitor is competitive or non-competitive. As the graph below shows, the presence of a competitive inhibitor does not change Vmax, but it does increase Km, indicating weaker affinity between the enzyme and the substrate. This weaker affinity is because there is direct competition between the inhibitor and the substrate to bind to the same active site. A larger concentration of substrate is required in order to have the same enzyme activity level.

For a non-competitive inhibitor, the overall enzyme activity is much lower, since the inhibitor is binding to the allosteric site, changing the shape of the active site and not allowing the substrate to bind as efficiently. As the graph below shows, the presence of a non-competitive inhibitor lowers Vmax, but it does not change Km, indicating the same level of affinity between the enzyme and the substrate, as there is no direct competition for binding between the inhibitor and the substrate.

Summary

Inhibitors in metabolic pathways

Many metabolic processes have feedback loop systems that allow for control over the rate of production of the end product, being dependent on the concentration of the end product. These feedback systems can be 'positive', where the presence of the end product will result in an increased rate of the metabolic pathway, such as in the case of the hormone oxytocin during labour. A small quantity of oxytocin produced initially will set off a positive feedback loop to keep increasing the rate of production of oxytocin to keep labour progressing until the baby is born.

Other feedback loop systems are 'negative', that is, the presence of the end product results in a decreased rate of the metabolic pathway. For example, when sugar is consumed and the concentration of sugar rises in the blood, the hormone insulin is released, causing cells to absorb the sugar from the blood. When blood sugar concentration in the blood falls, insulin is no longer released.

Since metabolic pathways often require multi-step processes involving a series of reactions that each may be catalysed by different enzymes, the presence of inhibitors can play a role in one or more steps of the pathway, affecting the overall outcome of the metabolic process.