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 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 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.