Reversible enzyme inhibitors

Enzyme inhibitors are classified in several ways. The inhibitor may interact either reversibly or irreversibly with the enzyme. Reversible inhibitors interact with the enzyme through noncovalent association/dissociation reactions. In contrast, irreversible inhibitors usually cause stable, covalent alterations in the enzyme. That is, the consequence of irreversible inhibition is a decrease in the concentration of active enzyme. The kinetics observed are consistent with this interpretation, as we shall see later. 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

Impurities or the ions of the liquid themselves may act as reversible or irreversible enzyme inhibitors. 

The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows Michaelis-Menten kinetics and is given by kj(obs) = ki(max) [I]/(Ki + [1]), where Kj is the dissociation constant of an initially formed, non-covalent, enzyme-inhibitor complex which is converted into the covalent reaction product with the rate constant kj(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near Kj. In this case, only the second-order rate-constant kj(max)/Kj can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)]. In the presence of these compounds, the inactivation rate Kj(obs) should be diminished by an increase of Kj by the factor (1 + [S]/K, ) or (1 + [I(rev)]/I (rev)). From the dependence of kj(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine Kj for inhibitors that react too rapidly in the accessible range of concentration. ... 

As we have just seen, the initial encounter complex between an enzyme and its substrate is characterized by a reversible equilibrium between the binary complex and the free forms of enzyme and substrate. Hence the binary complex is stabilized through a variety of noncovalent interactions between the substrate and enzyme molecules. Likewise the majority of pharmacologically relevant enzyme inhibitors, which we will encounter in subsequent chapters, bind to their enzyme targets through a combination of noncovalent interactions. Some of the more important of these noncovalent forces for interactions between proteins (e.g., enzymes) and ligands (e.g., substrates, cofactors, and reversible inhibitors) include electrostatic interactions, hydrogen bonds, hydrophobic forces, and van der Waals forces (Copeland, 2000). 

In this chapter we described the thermodynamics of enzyme-inhibitor interactions and defined three potential modes of reversible binding of inhibitors to enzyme molecules. Competitive inhibitors bind to the free enzyme form in direct competition with substrate molecules. Noncompetitive inhibitors bind to both the free enzyme and to the ES complex or subsequent enzyme forms that are populated during catalysis. Uncompetitive inhibitors bind exclusively to the ES complex or to subsequent enzyme forms. We saw that one can distinguish among these inhibition modes by their effects on the apparent values of the steady state kinetic parameters Umax, Km, and VmdX/KM. We further saw that for bisubstrate reactions, the inhibition modality depends on the reaction mechanism used by the enzyme. Finally, we described how one may use the dissociation constant for inhibition (Kh o.K or both) to best evaluate the relative affinity of different inhibitors for ones target enzyme, and thus drive compound optimization through medicinal chemistry efforts. 

Table 5.1 Characteristic effects of substrate concentration on the IC50 value for reversible enzyme inhibitors of different modalities...

If the inhibition is found to be rapidly reversible, we must next determine if the approach to equilibrium for the enzyme-inhibitor complex is also rapid. As described in Chapter 4, some inhibitors bind slowly to their target enzymes, on a time scale that is long in comparision to the time scale of the reaction velocity measurement. The effect of such slow binding inhibition is to convert the linear progress curve seen in the absence of inhibitor to a curvilinear function (Figure 5.10). When nonlinear progress curves are observed in the presence of inhibitor, the analysis of... 

Not all enzyme inhibitors bind through reversible interactions. In some cases enzymes are inactivated by formation of covalent complexes with inhibitory molecules. 

Until now our discussions of enzyme inhibition have dealt with compounds that interact with binding pockets on the enzyme molecule through reversible forces. Hence inhibition by these compounds is always reversed by dissociation of the inhibitor from the binary enzyme-inhibitor complex. Even for very tight binding inhibitors, the interactions that stabilize the enzyme-inhibitor complex are mediated by reversible forces, and therefore the El complex has some, nonzero rate of dissociation—even if this rate is too slow to be experimentally measured. In this chapter we turn our attention to compounds that interact with an enzyme molecule in such a way as to permanendy ablate enzyme function. We refer to such compounds as enzyme inactivators to stress the mechanistic distinctions between these molecules and reversible enzyme inhibitors. 

However, the conversion of omeprazole to the active sulphenamide does not result in formation of a reversible enzyme inhibitor, but rather results in in situ formation of a powerful affinity label. Hence we can consider omeprazole to be a unique example of quiescent affinity labeling in which selectivity results from the unique environment of the target enzyme. 

More than 3000 different enzymes have been extracted from animals, plants and microorganisms. Traditionally, they have been used in impure form since purification is expensive and pure enzymes may be difficult to store and use. There is usually an optimum temperature and pH for maximum activity of an enzyme. Outside these optimum conditions, activity may simply be held in check or the enzyme may become denatured , i.e. altered in such a way that activity is lost permanently, although some forms of denaturing are reversible. Many enzymes are also sensitive to transition-metal ions, the effect being specific to particular metal ions and enzymes. In some cases, certain metal ions are essential for the stability and/or activity of an enzyme. In other cases, metal ions may inhibit the activity of an enzyme. Similarly, certain organic compounds can act as enzyme inhibitors or activators. 

The transition state analog (TSA) approach1651 which has proved so successful in the design of enzyme inhibitors and catalytic antibodies lends itself nicely, at least in principle, to the molecular imprinting of polymers. Polymerization carried out in the presence of the TSA, or with the TSA covalently but readily reversibly bound to a monomer, produces a polymer with a number of embedded TSA molecules. If these can be removed under rea-... 

Reversible inhibitors are more subtle and act usefully to control the rate of particular enzymes. Often, reversible inhibitors are substrates found at or near the end of a pathway. These compounds act in a negative feedback manner to slow down the activity of an enzyme at or near the beginning of the same pathway. Occasionally, feedback inhibitors may be substrates found within a pathway which is functionally related to the one in which the target enzyme can be found. Furthermore, the products of an enzyme-catalysed reaction are often inhibitory to the enzyme that generated them (Figure 3.2). This is should not be surprising from a structural point of view because the product must fit into the active site of the enzyme and so block the binding of substrate. 

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