Neuropathy Target Esterase of Hen Brain: Active Site Reactions with 2-[octyl-3H]Octyl-4H-1,3,2-Benzodioxaphosphorin 2-Oxide and 2-Octyl-4H-1,3,2-[aryl-3H] Benzodioxaphosphorin 2-Oxide

Abstract: 2-Octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide (octyl-BDPO) is one of the most potent inhibitors known for neuropathy target esterase (NTE) of hen brain with 50% inhibition at 0.2 nM. Two NTE-like proteins, i.e., resistant to paraoxon and sensitive to mipafox, of ～155 and ～119 kDa (designated NTE-155 and NTE-119, respectively) are labeled by octyl-³H]octyl-BDPO and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with aryl-³H]octyl-BDPO is only ～15% of that with octyl-³H]octyl-BDPO, indicating that the majority of the phosphorylated NTE undergoes aging with only a small proportion of nonaged target or intramolecular group transfer (“alkylation”). NTE-155 and NTE-119 have the same kinetic constants and maximal number of phosphorylation sites, equivalent for each of them to 26 fmol/mg of protein and totalling at least 0.44–1.2 µg of NTE protein/g of brain. Structure-activity investigations involving 17 combinations of organophosphorus (OP) compounds of varied chemical type, stereo-chemistry, and concentration establish an excellent correlation (r = 0.95) between inhibition of NTE activity and protein labeling and thereby the toxicological relevance of these assays, which equally implicate NTE-155 and NTE-119 (probably an autolysis product of NTE-155) as targets in OP-induced delayed neuropathy. octyl-³H]-Octyl-BDPO is an improved probe for NTE in terms of its potency, reactivity, selectivity, and the formation of ³H-labeled NTE with a stable phosphorus-carbon bond.