Resolving Pathways of Interaction of Covalent Inhibitors with the Active Site of Acetylcholinesterases: MALDI-TOF/MS Analysis of Various Nerve Agent Phosphyl Adducts

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• 作者：Eytan Elhanany ; Arie Ordentlich ; Or Dgany ; Dana Kaplan ; Yoffi Segall ; Ruth Barak ; Baruch Velan ; and Avigdor Shafferman
• 刊名：Chemical Research in Toxicology
• 出版年：2001
• 出版时间：July 2001
• 年：2001
• 卷：14
• 期：7
• 页码：912 - 918
• 全文大小：102K
• 年卷期：v.14,no.7(July 2001)
• ISSN：1520-5010

文摘

Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE withtoxic organophosphate compounds is both a biochemical and a pharmacological challenge. Herewe describe experiments which allowed to resolve some of the less well understood reactionpathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Trypticdigests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptidefractionation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the isotopically resolved peaks of organophosphoroamidate conjugates of the AChE active site Ser peptides. For tabun and its hexadeuterioanalogue, we find, as expected, that the two phosphoramidate adducts of the active site peptidediffer by 6.05 mass units but following aging we find that the two corresponding phospho-peptides have identical molecular weights. We further show that the aging product of paraoxon-AChE adduct is identical to the aging product of the tabun-AChE conjugate. These resultsunequivocally demonstrate that the pathway of aging of tabun adducts of the human or theTorpedo californica AChEs proceeds through P-N bond scission. For methamidophos, we showthat phosphylation of AChE involves elimination of the thiomethyl moiety and that thespontaneous reactivation of the resulting organophosphate adduct generates the phosphorusfree AChE active site Ser-peptide.