文摘
Aging of phosphylated serine esterases, e.g., acetylcholinesterase (AChE) and neuropathy target esterase(NTE), renders the inhibited enzymes refractory to reactivation. This process has been considered torequire postinhibitory side group loss from the organophosphorus moiety. Recently, however, it has beenshown that the catalytic domain of human NTE inhibited by N,N'-diisopropylphosphorodiamidofluoridate(mipafox, MIP) ages by deprotonation. For mechanistic understanding and biomarker development, itwould be important to know the identity of the MIP adduct on target esterases after inhibition and agingoccurred. Accordingly, the present study was performed to determine if MIP-inhibited human AChEages by side group loss or an alternate method, e.g., deprotonation. Diisopropylphosphorofluoridate (DFP),the oxygen analogue of MIP, was used for comparison, because DFP-inhibited AChE is known to ageby net loss of an isopropyl group. Kinetics experiments were done with DFP and MIP against AChE tofollow the time course of inhibition, reactivation, and aging for each inhibitor. MS studies of trypticdigests from kinetically aged DFP-inhibited AChE revealed a mass shift of 122.8 ± 0.7 Da for the activesite peptide (ASP) peak, corresponding to the expected monoisopropylphosphoryl adduct. In contrast,the analogous mass shift for kinetically aged MIP-inhibited AChE was 80.7 ± 0.9 Da, corresponding toa phosphate adduct. Because this finding was unexpected, the identity of the phosphoserine-containingASP was confirmed by immunoprecipitation followed by MS. The results indicate that aging of MIP-inhibited AChE proceeds by displacement of both isopropylamine groups. Further research will be requiredto elucidate the detailed mechanism of formation of a phosphate conjugate from MIP-inhibited AChE;however, knowledge of the identity of this adduct will be useful in biomarker studies.
