Contribution of Aromatic Moieties of Tyrosine 133 and of the Anionic Subsite Tryptophan 86 to Catalytic Efficiency and Allosteric Modulation of Acetylcholinesterase

Abstract

Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.

The Architecture of Human Acetylcholinesterase Active Center Probed by Interactions with Selected Organophosphate Inhibitors

“…These residues include Tyr-337 and Phe-338 (which are part of the hydrophobic alkoxy site); Trp-86 (the "anionic" subsite (Ordentlich et al, 1993a(Ordentlich et al, , 1995; Glu-450, Glu-202, and Tyr-133 key elements maintaining the functional architecture of the active center (Ordentlich et al, 1993b(Ordentlich et al, , 1995; and Phe-295 and Phe-297, which confer specificity of the acyl pocket (Fournier et al, 1992;Harel et al, 1992;Vellom et al, 1993;Ordentlich et al, 1993a;Gnatt et al, 1994).…”

“…Its catalytic power and the high reactivity toward organophosphorus inhibitors are believed to be determined by the unique architecture of the AChE active center, consisting of several subsites. Resolution of the three-dimensional structure of Torpedo AChE (Sussman et al, 1991), sitedirected mutagenesis, and molecular modeling together with kinetic studies of the AChE muteins with substrates and reversible inhibitors (Gibney et al, 1990;Velan et al, 1991aVelan et al, , 1991bShafferman et al, 1992aShafferman et al, , 1992bShafferman et al, , 1992cShafferman et al, , 1993Ordentlich et al, 1993aOrdentlich et al, , 1993bOrdentlich et al, , 1995Radic et al, 1992Radic et al, , 1993Barak et al, 1994;Kronman et al, 1994;Gnatt et al, 1994) (for a recent review see also Taylor and Radic (1994)) are beginning to unveil the functional role of the various active center subsites in the reactivity characteristics of the enzyme: (a) the esteratic site containing the active site serine; (b) the "anionic subsite," 2 ; (c) the hydrophobic site for the alkoxy leaving group of the substrate including residues , , and Phe-338(331); and (d) the acyl pocket, and . Apart from the esteratic subsite, the main contribution of these active center components to the catalytic activity appears to be in the stabilization of the Michaelis-Menten complexes, since most of the structural perturbations of the active center hardly affect the rate of the nucleophilic step (Ordentlich et al, 1993a).…”

Genetic and Epigenetic Mechanisms Underlying Acute and Delayed Neurodegenerative Consequences of Stress and Anticholinesterase Exposure

“…Inhibition of AChE activity values were determined from triplicate assays. IC50 values were calculated by Grafit (version 3, Erithacus Software, Staines, UK) and K; values were then calculated by the method of Hobbiger and Peck (1969), based the K m value of human AChE for ATCh of 0.14 mM (Ordentlich, et al, 1995). In the case of irreversible inhibitors, both kcat and K, were determined by adsorbing the chosen enzyme to antibody-coated wells of microtiter plates, exposing them to an inhibitor at several concentrations and for varying lengths of time, washing to remove the inhibitor, and assaying for remaining activity.…”

Overlay-independent comparisons of X-ray structures reveal small, systematic conformational changes in liganded acetylcholinesterase

“…The magnitude of the C terminal alpha helix motions is much larger for the POX-hAChE conjugate. Consistent exceptions in both structures are expanding positions of Glu and His of the catalytic triad and a small loop around Tyr133, a residue known to protrude into the choline binding site of the active center with hydrogen bonding capacity (8). The clear and significant difference between two covalent conjugates is in the acyl pocket where sharp, blue 1.2 Å positive "Δ distance" peak is observed at the position 296 of POX-hAChE, combined with smaller, 0.26 Å expansion of the peripheral site Trp286 and opposed by 0.8-0.6 Å contractions at positions 291 and 292 of the acyl pocket.…”