Energetics of ligand and inhibitor interactions with acetylcholinesterase

Abstract

Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus, purified by affinity chromatography to a specific activity of 7000 – 10000 U/mg protein, was studied at 27 °C in conduction-type microcalorimeters for the heats of reaction, with the subsite-specific cationic ligands edrophonium and propidium and with the irreversible inhibitor diisopropylfluorophosphate (DFP), in an ion-free aqueous medium. Edrophonium and propidium, each at 0.5 × 10−5 M, yielded reaction heats of +3.2 and –1.5 kcal/mol (1 kcal = 4.184 J) respectively, with 1.3 × 10−5 M AChE active sites. DFP (1.3 × 10−5 M) reacted exothermically yielding −0.5 kcal/mol at stoichiometric level with AchE active sites. Circular dichroic spectra showed that a ternary complex of AChE (6.5 × 10−7 M active sites) and the two ligands (each at 1 × 10−3 M) in 1 mM Tris–HCl buffer (pH 8.0) had a positive Cotton effect at 235 nm. Neither DFP nor phosphoric acid 2,2-dichloroethenyl dimethyl ester (DDVP) caused any appreciable change. DFP–AChE, however, behaved like a normal enzyme in showing a positive Cotton effect in association with the two ligands. DDVP–AChE showed an increase in negative ellipticity at 287 nm in the presence of the two ligands. Another cationic ligand, d-tubocurarine, when present together with edrophonium, increased negative ellipticity at 302 nm and blue-shifted a 265-nm peak of the normal AChE. DFP interactions with AChE appear to be energetically different from those of edrophonium, the latter of which is believed to associate with the acetylcholine-binding subsite.