Allosteric Modulation of Acetylcholinesterase Activity by Peripheral Ligands Involves a Conformational Transition of the Anionic Subsite

Abstract

Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.

X-Ray Crystallographic Studies on Acetylcholinesterase and Related Enzymes

“…This residue is strategically placed near the top of the active-site gorge (Fig. 1) and was identified as a crucial component of the PAS Barak et al, 1995). Strong evidence in support of our model comes from the observation that in mutant enzymes in which the putative cationic trap has been removed by neutralizing the charge on D72, on-rate values for cationic ligands fall to the theoretical values for neutral ones ( Table 2).…”

“…The importance of cation-7T interactions in the catalytic function of ChEs has been confirmed by a large body of structural Modeling of the D72Y mutant of TcAChE shows that the hydroxyl moiety of the tyrosine at position 72 is capable of forming a hydrogen bond with Y121. It has been hypothesized that the hydrogen bond between Y121 and D72 in WT AChE is required for maintaining the "functional crosstalk" postulated for the transduction of allosteric signals from the PAS to the catalytic center Barak et al, 1995). These considerations also provide us with a rather straightforward test for the presence of such crosswalk, because replacing tyrosine with phenylalanine, i.e., generating the D72F mutant, should have a disruptive effect on signal transduction and reduce catalytic efficiency significantly.…”

“…During development of the nervous system of vertebrates, expression of ChEs is associated with periods of enhanced neurite outgrowth Layer et al, 1993), suggesting that they might serve as neuronal growth factors or cell-adhesion molecules. It has been speculated that the site responsible for the non-catalytic interaction of ChEs is localized close to the entrance of the active-site gorge, perhaps overlapping with what has been termed the 'peripheral' anionic site (PAS) of AChE, a possible locus of regulation of enzymatic activity Eichler et al, 1994;Barak et al, 1995), which is located in the region of the 'annular' electrostatic motif. In a recent study, AChE was shown to promote neurite regeneration in cultured adult neurons of the mollusk Aplysia .…”

“…Solution of the three-dimensional (3D) structure of AChE from Torpedo californica (7cAChE) was followed by determination of the cry stak structures of several complexes of AChE with specific inhibitors . Analysis of these structures, taken together with systematic site-directed mutagenesis studies Barak et al, 1995), has permitted identification of the key functional residues in the active site and contributed to clarification of their role in recognition of substrates and inhibitors. (A comprehensive and updated list of references for cholinesterase mutants can be obtained through the ESTHER server, http://meleze.ensam.inra.fr/cholinesterase (Cousin et al, 1997).)…”

Site‐directed mutants designed to test back‐door hypotheses of acetylcholinesterase function

“…mutant, G80C/V431C, in which the two cysteines were linked It has been suggested that binding of a 'peripheral' site by a disulfide bridge, led to a reasonable structure after a few ligand, at the top of the active-site gorge, might produce a cycles of energy minimization (Fig. 1). conformational transition within the 'anionic' subsite of the Based on the above considerations, a double mutant was catalytic site, specifically in the ~ loop containing Trp s4 [14]. …”