Organophosphate inhibitors: the reactions of bis(p-nitrophenyl) methyl phosphate with liver carboxylesterases and alpha-chymotrypsin.

Bis(p-nitrophenyl) methyl phosphate (BNMP) has been tested as a spectrophotometric titrant for a group of serine hydrolases. Bis(p-nitrophenyl) methyl phosphate reacts rapidly with liver carboxylesterases from chicken, sheep, and horse, and more slowly with alpha-chymotrypsin, releasing 2 mol of p-nitrophenol per active site titrated, and producing a phosphorylated enzyme very stable to dephosphorylation. However, pig liver carboxylesterase produces 2.2 mol of p-nitrophenol per active site titratedmreaction of pig and chicken liver carboxylesterases with bis(p-nitrophenyl) [3H]methyl [32P]phosphate clarified this differencemone molecule of the chicken enzyme reacts with one molecule of bis(p-nitrophenyl) methyl phosphate, releasing both p-nitrophenol residues, and resulting in an inhibited enzyme with one phosphorus atom and one methyl group covalently bound. Pig enzyme reacts rapidly, forming (presumably) methyl p-nitrophenyl phosphoryl-carboxylesterasemthis further reacts, concurrently producing methyl phosphoryl-carboxylesterase plus p-nitrophenol, or free enzyme plus methyl p-nitrophenyl phosphate, in the ratio of about 5 : 1 at pH 7.55. The free enzyme produced undergoes further reaction with bis(p-nitrophenyl) methyl phosphate until all the carboxylesterase is inhibited.