Tight-binding inhibitors, reversible inhibition

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. 

In addition to its broad-spectrum biocidal activity, triclosan (22) displays reversible inhibition of E. coli Fabl with a picomolar K, for binding the enzyme-cofactor complex [4]. Triclosan entry results in the reordering of a loop of amino acids close to the active site, making it a slow, tight-binding inhibitor [41]. 

Irreversible enzyme inhibition, also cahed enzyme inactivation (or active-site directed ineversible inhibition, because it is generally competitive with substrate), occurs when a compound blocks the enzyme activity for an extended period of time, generally via covalent bond formation. Therefore, even though some slow tight-binding inhibitors functionahy block the enzyme activity irreversibly, they are stih considered reversible... 

Sodium l,l-dimethoxyethyl(methyl)phosphinate 2 was found to be the most effective herbicidal compound among plant PDHc El inhibitors by Baillie et al. s work. 2 was presumably hydrolyzed to sodium salt of acetyl(methyl)phosphinic acid 1-2 in vivo to exhibit herbicidal activity (Scheme 4.10). 1-2 displayed higher enzyme inhibition and herbicidal activity than 1-1. It has been found that 1-1 was a competitive inhibitor of PDHc, but 1-2 caused time-dependent inhibition. Baillie et al. gave a possible explanation for this result, the initial binding of inhibitors to the pyruvate site and subsequent reaction with thiamine pyrophosphate were rapid and reversible for both 1-1 and 1-2. In the case of 1-2, an enzyme-inhibitor complex was first formed and then underwent a time-dependent, essentially irreversible transformation to produce a more tightly bound form. In other words, 1-2 could act as a slow, tight binding inhibitor [1]. 

Szedlacsek, S. E., Ostafe, V., Duggleby, R. G., Serban, M., Vlad, M. O. (1990). Rrogress-Curve Equations for Reversible Enzyme-Catalyzed Reactions Inhibited by Tight-Binding Inhibitors. Biochem. J. 265,647-653. 

A (rapidly) reversible inhibitor will permit rapid and complete recovery of enzyme activity by dialysis. However, irreversible inhibitors are not removed by this procedure. Recovery from tight-binding inhibition is usually slow it is not uncommon for several dialysis bags containing enzyme to be prepared and for activity in each to be determined at various time points following the commencement of dialysis. The off-rate of these inhibitors is generally more rapid at higher temperatures. 

A number of conformationally restricted fluorinated inhibitors have been synthesized and evaluated. These smdies show that (1) subtle conformational differences of the substrates affect the inhibition (potency, reversible or irreversible character) (Figure 7.50), (2) a third inhibition process involving an aromatization mechanism could take place (Figure 7.51). When the Michael addition and enamine pathways lead to a covalently modified active site residue, the aromatization pathway produces a modified coenzyme able to produce a tight binding complex with the enzyme, responsible for the inhibition (Figure 7.51). ... 

Keller and coworkers341 proposed that tunicamyein is a reversible, tight-binding, and, therefore, competitive inhibitor of the GlcNAc 1-P transferase. The association rate-constant was 7 x 104 M s 1 (at 23°). Inhibition can be overcome by increasing the proportion of enzyme, and, because preincubation of the enzyme with UDP-GlcNAc prevented inhibition by tunicamyein,341 some experimental support for competitive inhibition was obtained. The known affinity of the antibiotic for phosphonolipids323 may facilitate its access to the membrane-bound enzyme, but the lipids do not prevent inhibition of the enzyme by tunicamyein.340... 

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent inhibitors provide a means of mapping the enzyme s active site. In contrast, reversible inhibition is characterized by a rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the substrate concentration. 

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