Inhibition of acetylcholinesterase by (1S,3S)-isomalathion proceeds with loss of thiomethyl: Kinetic and mass spectral evidence for an unexpected primary leaving group

Previous work demonstrated kinetically that inhibition of mammalian acetylc holinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethy l instead of the expected diethyl thiosuccinate as the primary leaving grou p followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation aft er inhibition with the (1S,3S)-stereoisomer. Electric eel acetylcholinester ase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k(3)) were measured. Oxime-mediated ha values were > 25-fold higher for enzyme inhibi ted by (1R)- versus (1S)-stereoisomers with the greatest contrast between t he (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (LR,3R)isomalath ion reactivated spontaneously and in the presence of pyridine-2-aldoxime me thiodide (S-PAM) with ha values of 1.88 x 10(5) and 4.18 x 105 min(-1), res pectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spon taneous and 2-PAM-mediated k(3) values of 0 and 6.05 x 10(3) min(-1), respe ctively. The kinetic data that were measured were consistent with those obt ained for mammalian AChE used in previous studies. Identification of the ad duct that renders EEAChE stable toward reactivation after inhibition with ( 1S,3S)-isomalathion was accomplished using a peptide mass mapping approach with matrix-assisted laser desorption/ionization time-of-night mass spectro metry (MALDI-TOF-MS). A peak with a mass corresponding to the active site p eptide containing the catalytic Ser with a covalently bound O-methyl phosph ate adduct was found in the mass spectra of (1S,3S)-treated EEAChE but not control samples. Identities of the modified active site peptide and adduct were confirmed by fragmentation in MALDI-TOF-MS post-source decay (PSD) ana lysis, and peaks corresponding to the loss of an adduct as phosphorous/phos phoric acid methyl ester were observed. The results demonstrate that inhibi tion of EEAChE by ( 1S,3S)-isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosucci nate as the secondary leaving group to yield an aged enzyme.