Slow-tight-binding enzyme inhibitors

Slow, tight-binding inhibition occurs when slow-binding inhibition takes place at inhibitor concentrations comparable to that of the enzyme, in which case the previous two mechanisms can still apply. Comprehensive review articles on the subject of tight, slow, and slow, tight-binding inhibitors ate available in the literature (12,14). 

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]. 

Some inhibitors interact very slowly with the enzyme protein, and onset of inhibition thus exhibits time-dependence. These inhibitors are generally referred to as slow-binding inhibitors, and as slow tight-binding inhihitors if the potency of inhibition is extremely high. Analysis of these inhibitory mechanisms is complex because binding and dissociation rate constants may be determined in addition to values. Indeed, a complete analysis may require extensive use of specialized computer software, and the complexities of such analyses preclude their discussion in this chapter. However, the reader is directed to several publications from Morrison s laboratory if a slow-binding mechanism is suspected for an inhibitor of interest (Morrison, 1982 Morrison and Stone, 1985 Sculley and Morrison, 1986 Morrison and Walsh, 1988). 

Morrison JF, Stone SR. 1985. Approaches to the study and analysis of the inhibition of enzymes by slow- and tight-binding inhibitors. Comments Mol Cell Biophys 2 347. Morrison JF, Walsh CT. 1988. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61 201. 

Classical NSAIDs and COX-2 inhibitors are time-dependent, irreversible inhibitors of hCOX-2, which is consistent with a two-step process, involving an initial rapid equilibrium binding of enzyme and inhibitor, followed by a slow formation of a tightly bound enzyme-inhibitor complex. COX-2 inhibitors show a time-independent inhibition of hCOX-1, consistent with the formation of a reversible enzyme-inhibitor complex (Ouellet and Percival 1995 Riendeau et al. 2001). 

Like IM and SU, TP is a slow, tight binding inhibitor with a much greater affinity for the steady state complex than the free enzyme. Mutants of tobacco and soybean resistant to TP have been isolated in tissue culture. Initial analysis suggests ALS in these mutants is desensitized to TP. 

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... 

For slow-tight-binding inhibitors, is very small and formation of the E. I complex is essentially irreversible. Use of Equation 17.28 ensures that depletion of free enzyme and free inhibitor by formation of the E. I complex is taken into account. 

To observe the slow onset of inhibition and the E. I complex, iiTj must be smaller than and smaller than 4. However, if is considerably smaller than 4, then the formation of the E. I complex will be effectively irreversible (i.e., the inhibitor is of the slow-tight-binding variety). Under those circumstances it will again be necessary to take depletion of free enzyme and free inhibitor into account when determining iiTi and K (78). 

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]. 

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