Miller_2025_Chem.Res.Toxicol__

Organophosphorus (OP) compounds pose a serious risk to human health by covalently modifying acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Currently approved oxime therapeutics can reactivate OP-inhibited AChE and BChE, despite significant limitations. The OP-inhibited enzymes undergo a secondary O-dealkylation event, known as aging, for which no currently approved therapeutics are effective as treatments. Many decades of research have studied the aging mechanism in AChE and BChE. It has previously been accepted that aging occurs via a spontaneous O-dealkylation event, by loss of a carbocation or by water hydrolysis of the OP-adducted serine residue. Here, we present a novel mechanism of aging in which the catalytic histidine acts as a nucleophile to induce aging and, as a result, becomes alkylated after exposure to methyl paraoxon (MP) and other pesticides. Using bottom-up proteomics, we identify that upon aging of MP-inhibited AChE and BChE, a methyl transfer occurs from the phosphylated serine residue to the catalytic histidine residue. The extent of histidine methylation is pH-dependent as less methylation is observed at lower pH, while increased methylation is observed at higher pH. At near physiological pH (7.5), the ratio of N-MeHis/His is 3:1 for AChE and 1.3:1 for BChE after 24 h. When other OP compounds were also tested for histidine modification, ethyl paraoxon was shown to result in ethylation of the catalytic histidine; however, when the alkoxy group was branched in the case of an isopropoxy group present in diisopropyl fluorophosphate, no alkylation of histidine was observed. Recent advances in the development of quinone methide precursors show promise in the recovery of OP-aged AChE. In this work, we discuss the importance of this novel aging mechanism and its impact on the recoverability of OP-aged AChE or BChE as it appears that the histidine modification limits the overall recovery of active AChE.