Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases: MALDI-TOF/MS analysis of various nerve agent phosphyl adducts

被引:69
作者
Elhanany, E
Ordentlich, A
Dgany, O
Kaplan, D
Segall, Y
Barak, R
Velan, B
Shafferman, A [1 ]
机构
[1] Israel Inst Biol Res, Dept Biochem & Mol Genet, IL-74100 Ness Ziona, Israel
[2] Israel Inst Biol Res, Dept Organ Chem, IL-74100 Ness Ziona, Israel
[3] Israel Inst Biol Res, Dept Analyt Chem, IL-74100 Ness Ziona, Israel
关键词
D O I
10.1021/tx0100542
中图分类号
R914 [药物化学];
学科分类号
100701 ;
摘要
Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a pharmacological challenge. Here we describe experiments which allowed to resolve some of the less well understood reaction pathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Tryptic digests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptide fractionation 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 hexadeuterio analogue, we find, as expected, that the two phosphoramidate adducts of the active site peptide differ by 6.05 mass units but following aging we find that the two corresponding phosphopeptides 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 results unequivocally demonstrate that the pathway of aging of tabun adducts of the human or the Torpedo californica AChEs proceeds through P-N bond scission. For methamidophos, we show that phosphylation of AChE involves elimination of the thiomethyl moiety and that the spontaneous reactivation of the resulting organophosphate adduct generates the phosphorus free AChE active site Ser-peptide.
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页码:912 / 918
页数:7
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