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

Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a p harmacological challenge. Here we describe experiments which allowed to res olve some of the less well understood reaction pathways of phosphylation an d "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N a gents) such as tabun or the widely used pesticide methamidophos. Tryptic di gests of phosphylated AChEs (from human and Torpedo californica), ZipTip pe ptide fractionation and matrix-assisted laser desorption ionization mass sp ectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the iso topically resolved peaks of organophosphoroamidate conjugates of the AChE a ctive site Ser peptides. For tabun and its hexadeuterio analogue, we find, as expected, that the two phosphoramidate adducts of the active site peptid e differ by 6.05 mass units but following aging we find that the two corres ponding phosphopeptides have identical molecular weights. We further show t hat the aging product of paraoxon-AChE adduct is identical to the aging pro duct of the tabun-AChE conjugate. These results unequivocally demonstrate t hat the pathway of aging of tabun adducts of the human or the Torpedo calif ornica AChEs proceeds through P-N bond scission. For methamidophos, we show that phosphylation of AChE involves elimination of the thiomethyl moiety a nd that the spontaneous reactivation of the resulting organophosphate adduc t generates the phosphorus free AChE active site Ser-peptide.