Inhibitor-enzyme complex

In the case of competitive inhibition, the equilibrium between the enzyme, E, the inhibitor, 1, and the enzyme-inhibitor complex, El, is described by the equilibrium constant Ki. 

This class of inhibitors usually acts irreversibly by permanently blocking the active site of an enzyme upon covalent bond formation with an amino acid residue. Very tight-binding, noncovalent inhibitors often also act in an irreversible fashion with half-Hves of the enzyme-inhibitor complex on the order of days or weeks. At these limits, distinction between covalent and noncovalent becomes functionally irrelevant. The mode of inactivation of this class of inhibitors can be divided into two phases the inhibitors first bind to the enzyme in a noncovalent fashion, and then undergo subsequent covalent bond formation. 

Usually, a rapid binding step of the inhibitor I to the enzyme E leads to the formation of the initial noncovalent enzyme-inhibitor complex E-I. This is usually followed by a rate determining catalytic step, leading to the formation of a highly reactive species [E—I ]. This species can either undergo reaction with an active site amino acid residue of the enzyme to form the covalent enzyme-inhibitor adduct E—I", or be released into the medium to form product P and free active enzyme E. 

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

A significant difference between pseudoirreversible inhibitors and mechanism-based inactivators is the reversibiUty of the inactivation. A complete evaluation of the mechanism involved would require evidence not only for the covalent enzyme-inhibitor complex, but also for its decomposition products and its rate of reactivation. It is often difficult to identify the active site amino acid residue covalently linked to the inhibitor because of the instabiUty of the complex. 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

The Ki value is the dissociation constant of an enzyme-inhibitor complex. If [E] and [I] are the concentrations of enzyme and its inhibitor and [El] is the concentration of the enzyme-inhibitor complex, there is an equilibrium of complex formation and detachment as follows ... 

Inhibition of Glycosidases by AIdono-l,S-lactones and Aldohexoses Expressed by the Dissociation Constant K of the Enzyme-Inhibitor Complex... 

A case similar to the slow, practically irreversible inhibition of jack bean a-D-mannosidase by swainsonine is represented by the interaction of castanospermine with isomaltase and rat-intestinal sucrase. Whereas the association constants for the formation of the enzyme-inhibitor complex were similar to those of other slow-binding glycosidase inhibitors (6.5 10 and 0.3 10 M s for sucrase and isomaltase, respectively), the dissociation constant of the enzyme-inhibitor complex was extremely low (3.6 10 s for sucrase) or could not be measured at all (isomaltase), resulting in a virtually irreversible inhibition. Danzin and Ehrhard discussed the strong binding of castanospermine in terms of the similarity of the protonated inhibitor to a D-glucosyl oxocarbenium ion transition-state, but were unable to give an explanation for the extremely slow dissociation of the enzyme-inhibitor complex. 

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. ... 

In noncompetitive inhibition, binding of the inhibitor does not affect binding of substrate. Formation of both EI and EIS complexes is therefore possible. However, while the enzyme-inhibitor complex can still bind substrate, its efficiency at transforming substrate to product, reflected by is decreased. Noncompetitive... 

Vijayalakshmi, J. Meyer, E. F. Kam, C.-M. Powers, J. C. Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivable doubly covalent enzyme-inhibitor complex. Biochemistry 1991, 30, 2175-2183. 

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... 

This approach is not restricted to bacterial or viral cells. Mammalian cells under highly proliferating conditions can be cultured at increasing exposure to a compound in attempts to create resistant mutants. Alternatively, one can sometimes use a structural biology approach to predict amino acid changes that would abrogate inhibitor affinity from study of enzyme-inhibitor complex crystal structures. If the recombinant mutant enzyme displays the diminished inhibitor potency expected, one can then devise ways of expressing the mutant enzyme in a cell type of interest and look to see if the cellular phenotype is likewise abolished by the mutation. 

For the enzyme isomerization mechanism illustrated in scheme C of Figure 6.3, there are two steps involved in formation of the final enzyme-inhibitor complex an initial encounter complex that forms under rapid equilibrium conditions and the slower subsequent isomerization of the enzyme leading to the high-affinity complex. The value of kohs for this mechanism is a saturable function of [/], conforming to the following equation ... 

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. 

As an illustration of first-order kinetics, let us consider the simple dissociation of a binary enzyme-inhibitor complex ( 7) to the free enzyme (E) and the free inhibitor (/),... 

Inhibition Effects in Enzyme Catalyzed Reactions. Enzyme catalyzed reactions are often retarded or inhibited by the presence of species that do not participate in the reaction in question as well as by the products of the reaction. In some cases the reactants themselves can act as inhibitors. Inhibition usually results from the formation of various enzyme-inhibitor complexes, a situation that decreases the amount of enzyme available for the normal reaction sequence. The study of inhibition is important in the investigation of enzyme action. By determining what compounds behave as inhibitors and what type of kinetic patterns are followed, it may be possible to draw important conclusions about the mechanism of an enzyme s action or the nature of its active site. 

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