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Enzymes are the key to most processes required for life. They catalyze reactions that reduce the energy needed to produce products.
However, enzymes are also susceptible to being inhibited. Inhibitors are chemicals that bind to an enzyme to prevent it from catalyzing a reaction. Some inhibitors are reversible, while others are irreversible.
Competitive Inhibition
Enzymes are responsible for a wide range of chemical reactions in the cells of the body. These reactions can be vital for life, but sometimes enzymes need to be turned off to prevent their activity from becoming harmful. For example, enzymes are used to form clots in the blood to stop blood loss, but these clots can also block the flow of oxygen and nutrients to other parts of the body. Therefore, enzymes have evolved mechanisms to be turned off. These mechanisms usually involve inhibitors, molecules that bind to an enzyme and prevent it from catalyzing its reaction.
Inhibitors can be divided into three groups: competitive, uncompetitive and mixed inhibitors. The difference between these groups lies in the way the inhibitors bind to an enzyme.
Competitive inhibition occurs when a substance competes for the same binding site on an enzyme with a substrate, altering both Michaelis-Menten kinetics and the slope of the Lineweaver-Burk plot. However, unlike the other types of inhibitors, this type of inhibition is reversible and can be reduced by increasing the concentration of substrate.
Substrates bind to an enzyme through what are called active sites, which are located at different parts of the enzyme. These are often weak, reversible bonds. In contrast, allosteric sites are a different location on an enzyme and usually involve more permanent, covalent bonds between the substrate and the enzyme.
As an inhibitor binds to the active site, it changes the way that the enzyme folds into its tertiary structure. This changes the shape of the enzyme’s active site, making it less likely for a substrate to attach to it.
Noncompetitive inhibition, on the other hand, does not change the enzyme’s active site and decreases its maximum rate without affecting the Michaelis constant or Km. This type of inhibition can be reversed by increasing the substrate concentration, but can also lead to increased unused substrates accumulating and competing with the inhibitor for the active site.
Inhibition can be experimentally determined by measuring the reaction rate at saturating substrate concentrations in the presence and absence of a fixed amount of an inhibitor. This can be done using a technique known as the Lineweaver-Burk method (Fig. 8.9A and B). In the absence of an inhibitor, Vmax is increased with low and very high substrate concentrations, while in the presence of an inhibitor, it decreases. This can be plotted in the presence of a double reciprocal representation to determine which mechanism is responsible for the change in activity.
The Lineweaver-Burk plot shows that the reaction rate increases with increasing inhibitor concentration in the absence of an inhibitor, while in the presence of an inhibitor, the Vmax decreases at all substrate concentrations. The inhibition constant, Ki, can be calculated from the secondary plots and Dixon plots.
A Lineweaver-Burk plot is an important technique for determining the kinetic parameters of an enzymatic reaction. It allows the optimum molar concentration of the inhibitor to be determined and provides information about how the enzyme’s inhibition constant, Ki, is affected by the amount of the inhibitor present.