Kinetic, equilibrium and spectroscopic studies on cation association at the active center of acetylcholinesterase: topographic distinction between trimethyl and trimethylammonium sites

Abstract

This study examines the importance of electrostatic interactions on ligand association at the active center of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). The active-center serine was covalently modified with the dimensionally equivalent isosteric beta-(trimethylammonium)ethyl and 3,3-dimethylbutyl methylphosphonofluoridates. Reactivation of the 3,3-dimethylbutyl methylphosphono-conjugate by the bisquaternary mono-oxime HI-6, after accounting for the capacity for spontaneous reactivation, proceeded at a rate that was 20-fold greater than that for the cationic conjugate. Decidium, a fluorescent bisquaternary ligand that binds with its trimethylammonium moiety within the active center, exhibited affinity for the 3,3-dimethylbutyl conjugate that was within 2-fold that for the native enzyme, but 100-fold greater than for the cationic conjugate. Whereas association of n-alkyl mono- and bisquaternary ligands with the uncharged conjugate was virtually unaltered with respect to the native enzyme, the affinities of edrophonium, phenyltrimethylammonium and N-methylacridinium were reduced 100-fold for the uncharged conjugate relative to native enzyme. These results indicate that the orientations of the 3,3-dimethylbutyl and beta-(trimethylammonium)ethyl moieties with respect to the surface of the enzyme are not equivalent, that modification of the active center does not preclude cation association of active-center-selective ligands, and that aromatic cations associate at an anionic locus which is unique from that at which decidium and the n-alkyl mono- and bisquaternary cations associate. As such, the results point to the presence of a heterogeneity of cation binding sites within a circumscribed distance from the modified serine, and do not sustain the view proposed by Hasan et al. (J. Biol. Chem. 255 (1980) 3898-3904; 256, (1981) 7781-7785) that electrostatic interactions at the active center are subordinate to steric constraints imposed by a dimensionally restricted trimethyl site.