Fasciculin, a 6750-Da peptide from the venom of Dendroaspis, is known to inhibit reversibly mammalian and fish acetylcholinesterases at picomolar concentrations, but is a relatively weak inhibitor of avian, reptile, and insect acetylcholinesterases and mammalian butyryl-cholinesterases. An examination of fasciculin association with several mutant forms of recombinant DNA-derived acetylcholinesterase from mouse shows that it interacts with a cluster of residues near the rim of the gorge on the enzyme. The aromatic residues, Trp286, Tyr72, and Tyr124, have the most marked influence on fasciculin binding, whereas Asp74, a charged residue in the vicinity of the binding site that affects the binding of low molecular weight inhibitors, has little influence on fasciculin binding. The 3 aromatic residues are unique to the susceptible acetylcholinesterases and, along with Asp74, constitute part of the peripheral anionic site. Fasciculin falls in the family of three-loop toxins that include the receptor blocking alpha-toxins and cardiotoxins. From this basic structural motif, a binding site has evolved on fasciculin to be highly specific for the peripheral site on acetylcholinesterase. Acetylthiocholine affects rates of fasciculin binding at concentrations causing substrate inhibition. In the case of the mutant cholinesterases where rates of fasciculin dissociation are more rapid, steady state kinetic parameters also show acetylthiocholine-fasciculin competition to be consistent with occupation at a peripheral or substrate inhibition site rather than the active center.
Title: Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors Radic Z, Pickering NA, Vellom DC, Camp S, Taylor P Ref: Biochemistry, 32:12074, 1993 : PubMed
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.
