Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors

Abstract

By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.

Abundant Tissue Butyrylcholinesterase and Its Possible Function in the Acetylcholinesterase Knockout Mouse

“…We find that monomeric Triton X-100 reversibly competes for substrate binding to the active site, thereby inhibiting the activity of BChE. Binding of Triton X-100 appears to require the large Kinetic constants were calculated with SigmaPlot using the substrate activation equation of Radic et al (1993). The b value reflects the efficiency with which the substrate-enzyme-substrate complex forms product.…”

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

“…Inhibition constants were measured from the ratio of dissociation and association rates ascertained by conventional mixing and stopped-flow instrumentation (7). The mAChE Trp286Ala mutant was expressed, sequence verified, and concentrated from the expression medium as described (21). Purified AChE from D. melanogaster was a gift from D. Fournier (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France).…”

“…Alternate portals for substrate and product access have been proposed (38); however, catalytic and inhibitor-binding parameters are influenced only by mutations in the gorge (21) and not in the vicinity of the putative additional portals (39). Rapid fluctuations giving rise to transient enlargements of the gorge appear critical (40).…”

Soluble monomeric acetylcholinesterase from mouse: Expression, purification, and crystallization in complex with fasciculin

“…It was poured into a Bio-Rad econo column, allowed to pack by sedimentation, then washed with the equilibrating buffer (50-100-fold the bed volume). The bound AChE was eluted with 100 mM decamethonium bromide (30 x K , , RadiC et al 1993) in the same buffer, at a low flow rate (1-1.5 mL h-'). Elution fractions were assayed for AChE activity with a 106-fold final dilution, which reduced the final decamethonium concentration to well below its K , for the mouse enzyme.…”