Journal article
Inhibition of Acetylcholinesterase by (1S,3S)-Isomalathion Proceeds with Loss of Thiomethyl: Kinetic and Mass Spectral Evidence for an Unexpected Primary Leaving Group
Chemical research in toxicology, Vol.13(12), pp.1313-1320
12/18/2000
DOI: 10.1021/tx000184v
PMID: 11123973
Abstract
Previous work demonstrated kinetically that inhibition of mammalian acetylcholinesterase (AChE) by (1S)-isomalathions may proceed by loss of thiomethyl instead of the expected diethyl thiosuccinate as the primary leaving group followed by one of four possible modes of rapid aging. This study sought to identify the adduct that renders AChE refractory toward reactivation after inhibition with the (1S,3S)-stereoisomer. Electric eel acetylcholinesterase (EEAChE) was inhibited with the four stereoisomers of isomalathion, and rate constants for spontaneous and oxime-mediated reactivation (k 3) were measured. Oxime-mediated k 3 values were >25-fold higher for enzyme inhibited by (1R)- versus (1S)-stereoisomers with the greatest contrast between the (1R,3R)- and (1S,3S)-enantiomers. EEAChE inactivated by (1R,3R)-isomalathion reactivated spontaneously and in the presence of pyridine-2-aldoxime methiodide (2-PAM) with k 3 values of 1.88 × 105 and 4.18 × 105 min-1, respectively. In contrast, enzyme treated with the (1S,3S)-enantiomer had spontaneous and 2-PAM-mediated k 3 values of 0 and 6.05 × 103 min-1, respectively. The kinetic data that were measured were consistent with those obtained for mammalian AChE used in previous studies. Identification of the adduct 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-flight mass spectrometry (MALDI-TOF-MS). A peak with a mass corresponding to the active site peptide containing the catalytic Ser with a covalently bound O-methyl phosphate 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) analysis, and peaks corresponding to the loss of an adduct as phosphorous/phosphoric acid methyl ester were observed. The results demonstrate that inhibition of EEAChE by (1S,3S)-isomalathion proceeds with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate as the secondary leaving group to yield an aged enzyme.
Details
- Title: Subtitle
- Inhibition of Acetylcholinesterase by (1S,3S)-Isomalathion Proceeds with Loss of Thiomethyl: Kinetic and Mass Spectral Evidence for an Unexpected Primary Leaving Group
- Creators
- Jonathan A DoornDouglas A GageMichael SchallTodd T TalleyCharles M ThompsonRudy J Richardson
- Resource Type
- Journal article
- Publication Details
- Chemical research in toxicology, Vol.13(12), pp.1313-1320
- Publisher
- American Chemical Society
- DOI
- 10.1021/tx000184v
- PMID
- 11123973
- ISSN
- 0893-228X
- eISSN
- 1520-5010
- Language
- English
- Date published
- 12/18/2000
- Academic Unit
- Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984070358902771
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