Types of Inhibition

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There are 3 main types of inhibition (competitive, noncompetitive, and uncompetitive) that are most commonly used to describe the binding of an inhibitor to a target enzyme. However, a complete analysis of the mechanism of action requires the scientist to also evaluate other potential inhibition events, including allosteric, partial, tight-binding, and time-dependent inhibition. A review of these types of inhibition is provided.

Competitive Inhibition

A competitive inhibitor binds only to free enzyme. Often this binding event occurs on the active site of the target, precisely where substrate also binds. Although this is the case for a majority of competitive inhibitors, it is a misleading oversimplification. It is more appropriate to state that the binding of a competitive inhibitor and the binding of substrate are mutually exclusive events. Figure 1 below provides illustrations of some possible mutually exclusive binding events.

Figure 1 – Examples of Competitive Inhibition where Substrate (S) and Inhibitor (I) binding events are mutually exclusive. (a) Classical model for competitive inhibition where S and I compete for the same precise region of the active site. (b) I does not bind to the active site, but sterically hinders S binding. (c) S and I binding sites are overlapping. (d) S and I share a common binding pocket on the enzyme. (e) I binding can result in a conformational change that prevents S binding (and vice versa). This was adapted from Segal, Enzyme Kinetics.

Despite the differences in binding to the free enzyme illustrated in Figure 1, all competitive inhibitors have the same effects on substrate binding and catalysis. A competitive inhibitor will raise the apparent KM value for its substrate with no change in the apparent Vmax value. As a result, it is often stated that competitive inhibition can be overcome, observed by an increase in the apparent KI value, at higher concentrations of substrate. This characteristic will have physiological consequences on the observed efficacy of drugs. As an enzyme’s reaction is inhibited by a competitive inhibitor, there is an increase in the local concentration of substrate. Without a mechanism to clear the substrate, a competitive inhibitor will lose potency. This is not the case for a noncompetitive inhibitor.

Noncompetitive Inhibition

A noncompetitive inhibitor binds equally well to both free enzyme and the enzyme-substrate complex. These binding events occur exclusively at a site distinct from the precise active site occupied by substrate. Figure 2 provides some illustrations of the more common noncompetitive binding events.

Figure 2 – Examples of Noncompetitive Inhibition where Inhibitor (I) binding occurs at a site distinct from the Substrate (S) binding site and the Catalytic center (c) of the active site. (a) In this model, the binding of S induces a conformational change to align the catalytic center near S for catalysis. However, when I binds at a separate site, the conformational change does not occur and enzyme activity is inhibited. (b) In this model, I can sterically hinder S binding and release. However, unlike Figure 1-B, I and S can occupy the enzyme at the same time. This was adapted from Segal, Enzyme Kinetics.

In contrast to a competitive inhibitor, a noncompetitive inhibitor will lower the apparent Vmax value, yet there is no effect on the apparent KM value for its substrate. Essentially, the KI of the inhibitor does not change as a function of the substrate concentration.

In some circumstances, a compound may have unequal affinity for both free enzyme and the enzyme-substrate complex. This mixture of competitive and noncompetitive phenotypes is called mixed inhibition.

Uncompetitive Inhibition

An uncompetitive inhibitor binds exclusively to the enzyme-substrate complex yielding an inactive enzyme-substrate-inhibitor complex. When encountered, the apparent Vmax value and the apparent KM value should both decrease. Despite their rarity in drug discovery programs, uncompetitive inhibitors could have dramatic physiological consequences. As the inhibitor decreases the enzyme activity, there is an increase in the local concentration of substrate. Without a mechanism to clear the buildup of substrate, the potency of the uncompetitive inhibitor will increase.

Figure 3 – An example of Uncompetitive Inhibition where Inhibitor (I) only binds in the presence of Substrate (S).

Figure 4 – Illustrations of data demonstrating Competitive, Noncompetitive, and Uncompetitive Inhibition. The circles represent those rates obtained without the addition of inhibitor. The triangles contained 0.5xKI of inhibitor, the diamonds contained 2.0xKI of inhibitor, and the squares contained 4.0xKI of inhibitor. The black circles depict the shifts in the apparent KM for each binding modality.

Allosteric Inhibition

An allosteric inhibitor decreases activity by binding to an allosteric site, other than or in addition to the active site on the target. This interaction is characterized by a conformational change in the target enzyme that is required for inhibition. These conformational changes can affect the formation of the usual enzyme-substrate active site complex, stabilization of the transition state, or reduce the ability to lower the activation energy of catalysis. Figure 1e and Figure 2a are classical examples of allosteric inhibition. As such, an allosteric inhibitor may display a competitive, noncompetitive, or uncompetitive phenotype with respect to substrate binding.

Partial Inhibition

Partial inhibition results from the formation of an enzyme-substrate-inhibitor complex that can generate product with less facility than the enzyme-substrate complex. This can be illustrated in Figure 2a. When “I” is a partial inhibitor bound in the enzyme-substrate-inhibitor complex, the catalytic center may retain some ability to align near the substrate and facilitate catalysis. As a consequence of these structural changes, partial inhibitors can also be allosteric inhibitors of enzyme activity. In direct contrast, full inhibition results in an enzyme-substrate-inhibitor complex where the catalytic center is not capable of aligning near the substrate for catalysis.

Tight-Binding Inhibition

In this type of inhibition, the population of free, soluble inhibitor is significantly depleted by the formation of the enzyme-inhibitor or enzyme-substrate-inhibitor complex. While tight-binding inhibitors can bind to the target enzyme in a competitive, noncompetitive, or uncompetitive manner with respect to substrate binding, they can display noncompetitive phenotypes. However, a tight-binding inhibitor typically binds with an apparent affinity (KI) near the concentration of enzyme (active sites) present in the biochemical assay.

Time-Dependent Inhibition

Time-dependent inhibitors bind slowly to the enzyme on the time scale of enzymatic turnover, and thus display a change in initial velocity with time. This has the effect of slowing the observed onset of inhibition. Time-dependent inhibitors also impede the observed recovery of enzyme activity following inhibition, resulting in slow koff values. As illustrated in Figure 5, these inhibitors typically yield nonlinear initial velocities and nonlinear recoveries of enzyme activity.

Figure 5 – Illustrations of time-dependent inhibition. (a) This graph depicts the decrease in the initial velocity (product formed vs time) observed for classical, rapid equilibrium inhibitor and a time-dependent inhibitor. The latter yields a nonlinear progress curve consistent with a slow kon value. (b) This graph depicts the recovery of enzyme activity (product formed vs time) following dilution of the enzyme-inhibitor complex with substrate. Dilutions of classical, rapid equilibrium inhibitor complexes recover full activity immediately after dilution. Dilutions of time-dependent inhibitor complexes recover enzyme activity more slowly, indicative of a compound with a slow koff value. Dilutions of irreversible inhibitor complexes maintain the enzyme-inhibitor complex after dilution.

Some time-dependent inhibitors covalently attach to the target enzyme. For those inhibitors, the koff value is zero and the inhibition is said to be irreversible. These are typically less attractive molecules, unless the formation of the covalent species is specific to the reaction mechanism of the enzyme. Some inhibitors are for all practical purposes irreversible, with very low koff values, despite their inability to covalently attach to the enzyme. This stands in direct contrast to rapid equilibrium, reversible inhibitors that bind to and release from the enzyme at rates that are rapid in comparison to the rate of enzyme turnover.

Interestingly, many successful therapeutic drugs are time-dependent inhibitors. For these inhibitors with slow koff values, the rate of release of inhibitor from the enzyme-inhibitor complex (recovery of enzyme activity) proceeds independent of the substrate concentration and the physiological mechanism to remove inhibitor. This makes time-dependent inhibition a very attractive and proven strategy for the discovery and development of drugs.