title: The mipafox-inhibited catalytic domain of human neuropathy target esterase ages by reversible proton loss.
abstract: Aging of organophosphorus (OP)-compound-inhibited neuropathy target esterase (NTE) is the critical event that initiates OP-compound-induced delayed neurotoxicity (OPIDN). Aging has classically been considered to involve side-group loss from phosphylated NTE, rendering the enzyme refractory to reactivation. N,N'-Diisopropylphosphorodiamidofluoridate (mipafox, MIP)-inhibited NTE has been thought to age quickly; however, it can be reactivated under acidic conditions. The present study was undertaken to determine whether MIP-inhibited human recombinant NTE esterase domain (NEST) ages classically by isopropylamine loss. Diisopropylphosphorofluoridate (DFP), the oxygen analogue of MIP, was used for comparison. Kinetic values for DFP against NEST were as follows: k(i) = 17 200 +/- 180 M(-1) min(-1); reactivation t(1/2) approximately 90 min at pH 8.0 and approximately 60 min at pH 5.2; k(4) = 0.108 +/- 0.041 min(-1) at pH 8.0 and 0.181 +/- 0.034 min(-1) at pH 5.2. Kinetic values for MIP against NEST were as follows: k(i) = 1880 +/- 61 M(-1) min(-1); reactivation t(1/2) = 0 min at pH 8.0 and approximately 60 min at pH 5.2; aging was complete at all time points tested at pH 8.0, but no aging occurred at pH 5.2. Mass spectrometry revealed a mass shift of 123.0 +/- 0.6 Da for the active site peptide peak of aged DFP-inhibited NEST, corresponding to a monoisopropyl phosphate adduct. In contrast, the analogous mass shift for aged MIP-inhibited NEST was 162.8 +/- 0.6 Da, corresponding to the intact N,N'-diisopropylphosphorodiamido adduct. Thus, MIP-inhibited NEST does not age by isopropylamine loss. However, because kinetically aged MIP-inhibited NEST yields an intact adduct capable of reversible deprotonation, aging could occur by proton loss. Indeed, MIP-inhibited NEST does not age at pH 5.2 but ages immediately and completely at pH 8.0. Therefore, we conclude that the MIP-NEST conjugate ages by deprotonation rather than classical side-group loss.
authors.affiliation: Toxicology Program, Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan 48109-2029, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kropp
authors.forename: Paul
authors.initials: P
authors.lastname: Glynn
authors.forename: Rudy J
authors.initials: RJ
authors.lastname: Richardson
mesh_terms: D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004793:Enzyme Reactivators / Q000737:chemistry; D006801:Humans; D007531:Isoflurophate / Q000031:analogs & derivatives* / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009410:Nerve Degeneration / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D009943:Organophosphorus Compounds; D010713:Phosphatidylcholines; D011522:Protons; D007531:Isoflurophate; C005238:mipafox; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase; C017251:1,2-oleoylphosphatidylcholine