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

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.