DIFFERENCES IN ACTIVE-SITE GORGE DIMENSIONS OF CHOLINESTERASES REVEALED BY BINDING OF INHIBITORS TO HUMAN BUTYRYLCHOLINESTERASE

Amino acid sequence alignments of cholinesterases revealed that 6 of 1 4 aromatic amino acid residues lining the active center gorge of acety lcholinesterase are replaced by aliphatic amino acid residues in butyr ylcholinesterase. The Y337(F330) in mammalian acetylcholinesterase, wh ich is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridi nes and one end of bisquaternary compounds such as BW284C51 and decame thonium, Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution, Inhi bition studies of (-)-huperzine A with human butyrylcholinesterase mut ants, where A328 (K-I = 194.6 mu M) was modified to either F (K-I = 0. 6 mu M, as in Torpedo acetylcholinesterase) or Y (K-I = 0.032 mu M, as in mammalian acetylcholinesterase), confirmed previous observations m ade with acetylcholinesterase mutants that this residue is important f or binding huperzine A, Inhibition studies of ethopropazine with butyr ylcholinesterase mutants, where A328 (K-I = 0.18 mu M) was modified to either F (K-I = 0.82 mu M) or Y (K-I = 0.28 mu M), suggested that A32 8 was not solely responsible for the selectivity of ethopropazine, Vol ume calculations for the active site gorge showed that the poor inhibi tory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to acc ommodate the bulky inhibitor molecule. The volume of the butyrylcholin esterase active site gorge is similar to 200 Angstrom(3) larger than t hat of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigi d-body refinement and molecular dynamics calculations.