Tight-binding inhibitors, reversible

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]. 

Leupeptin is a slow, tight-binding inhibitor of trypsin. Some peptide aldehydes are potent, reversible inhibitors of cysteine proteases forming hemith-ioacetals with the active site cysteine.1 Similarly, peptide nitriles form thioimidate adducts.h... 

J.L Seymour, R.N. Lindquist, M.S. Dennis, B. Moffat, D. Yansura, D. Reiix M.E. Wessinger R.A. Lazarus, Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor-Xa. Biochemistry 1994, 33, 3949-3958. 

Most commercial HPPD inhibitors e.g., sulcotrione and isoxaflutole) are competitive time-dependent (tight-binding) inhibitors. As such, these herbicides bind to the enzyme very tightly with T A of dissociation ranging from a few hoins to several days, as opposed to milliseconds for traditional reversible inhibitors. Sorgoleone does not behave as these herbicides and appears to be a reversible inhibitor of HPPD. This quinone is structurally more planar than the traditional HPPD inhibitors, so it may not form a stable reaction intermediate. Instead, its backbone may resemble the conformation of one of the later intermediate step in the reaction mechanism of HPPD. 

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... 

In a recent report, by making a slight modification in the structure of the parent compound, two additional compounds with slightly higher activity have been synthesized (36). TFT and the aliphatic trifluoromethyl ketones appeared to be classical competitive inhibitors, while many trifluoropropanone sulfides were found to be reversible but slow and tight binding inhibitors of JHE... 

Many of the known inhibitors of serine and cysteine proteases feature the same types of war-head (e.g. oc-ketoamide, 8-lactam, aldehyde) which are able to undergo a covalent interaction with the nucleophilic active site alcohol or thiol group [371-373]. Depending on the reactivity of the war-head this approach has led to irreversible inhibitors or reversible but tight-binding inhibitors. The problem with this approach is that it is difficult to achieve selectivity and so there is considerable interest in the discovery of inhibitors that bind in a non-covalent fashion to the active site. 

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. 

---