Aging of Mipafox-Inhibited Human Acetylcholinesterase Proceeds by Displacement of Both Isopropylamine Groups to Yield a Phosphate Adduct Timothy J. Kropp and Rudy J. Richardson* Toxicology Program, Department of EnVironmental Health Sciences, UniVersity of Michigan, 1420 Washington Heights, Ann Arbor, Michigan 48109-2029 ReceiVed December 4, 2005 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 to require postinhibitory side group loss from the organophosphorus moiety. Recently, however, it has been shown that the catalytic domain of human NTE inhibited by N,N′-diisopropylphosphorodiamidofluoridate (mipafox, MIP) ages by deprotonation. For mechanistic understanding and biomarker development, it would be important to know the identity of the MIP adduct on target esterases after inhibition and aging occurred. Accordingly, the present study was performed to determine if MIP-inhibited human AChE ages 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 age by net loss of an isopropyl group. Kinetics experiments were done with DFP and MIP against AChE to follow the time course of inhibition, reactivation, and aging for each inhibitor. MS studies of tryptic digests from kinetically aged DFP-inhibited AChE revealed a mass shift of 122.8 ( 0.7 Da for the active site 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 to a phosphate adduct. Because this finding was unexpected, the identity of the phosphoserine-containing ASP 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 required to 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. Introduction Certain organophosphorus (OP) 1 compounds inhibit acetyl- cholinesterase (AChE), which leads to cholinergic toxicity in the central and peripheral nervous systems (1). Inhibition of AChE by OP compounds occurs by organophosphylation of the AChE active site Ser residue. The hydroxyl oxygen of the active site Ser attacks the OP compound at the phosphorus atom, displacing the primary leaving group, forming a covalent bond with the phosphyl moiety. The activity can be restored only by cleavage of the Ser-phosphyl bond and can be either spontane- ous (using water as the nucleophile) or mediated by other nucleophiles, such as the oxime, pyridine-2-aldoxime methiodide (2-PAM), or fluoride ion. Reactivation proceeds by S N 2 displacement of the enzyme from the phosphyl moiety, regen- erating enzymatically active AChE. However, the phosphylated serine may first undergo a postinhibitory aging process to yield an adduct that does not reactivate, either spontaneously or after oxime treatment. Aging is mechanistically defined as the creation of a negative charge on the phosphylated serine moiety. Aging usually involves scission of a side group from the phosphylated enzyme to yield the anionic species, although this may also occur by deprotonation (2). Aging of AChE inhibited by OP compounds of the phosphate and phosphonate classes has classically been thought to proceed by an S N 1 mechanism transiently yielding a carbocation (3, 4). Diisopropylphosphorofluoridate (DFP) is a thoroughly studied OP compound; the aging of DFP-inhibited enzymes has been shown to yield an anionic monosubstituted phosphyl ligand on the active site serine for AChE (5, 6), butyrylcholinesterase (BChE) (7), neuropathy target esterase (NTE) (5, 8, 9), and human recombinant NTE esterase domain (NEST) (2). N,N′- Diisopropylphosphorodiamidofluoridate (mipafox, MIP), the phosphoramidate analogue of DFP, is expected to interact with AChE in the same way as DFP does. MIP-inhibited AChE has been shown to be reactivated quickly in vitro by treatment with potassium fluoride after inhibition if reactivated within 18 h of inhibition (10). This suggests that conventional, time-dependent aging does occur for mipafox-inhibited enzymes in a manner similar to that for DFP-inhibited esterases. Furthermore, it is known that some N-alkyl phosphoramidates age by fission of the phosphorus-nitrogen bond (11, 12). The possibility of phosphorus-nitrogen bond fission suggests that the aging of mipafox-inhibited AChE may occur by the hydrolytic loss of an alkylamino group. However, the mechanism of mipafox- inhibited BChE or AChE aging needs to be investigated further; recent evidence has established that MIP-inhibited NEST ages by proton loss, yielding an intact N,N′-diisopropylphospho- * To whom correspondence should be addressed. Tel: 734-936-0769. Fax: 734-763-8095. E-mail: rjrich@umich.edu. 1 Abbreviations: 2-PAM, pyridine-2-aldoxime methiodide; AChE, ace- tylcholinesterase; ASP, active site peptide; ATCh, acetylthiocholine; BChE, butyrylcholinesterase; DFP, diisopropylphosphorofluoridate; DTNB 5,5′- dithio-bis(2-nitrobenzoic acid); MALDI, matrix-assisted laser desorption/ ionization; MH + , protonated molecule; MIP, N,N′-diisopropylphosphorodi- amidofluoridate, mipafox; NEST, human recombinant NTE esterase domain; NTE, neuropathy target esterase; OP, organophosphorus; SELDI, surface- enhanced laser desorption/ionization; TOF, time-of-flight. 334 Chem. Res. Toxicol. 2006, 19, 334-339 10.1021/tx050342o CCC: $33.50 © 2006 American Chemical Society Published on Web 01/04/2006