Evidence for P−N Bond Scission in Phosphoroamidate Nerve Agent Adducts of Human Acetylcholinesterase

• Published in: Biochemistry (Easton) Vol. 39; no. 5; pp. 1156 - 1161
• Main Authors: Barak, Dov, Ordentlich, Arie, Kaplan, Dana, Barak, Ruth, Mizrahi, Dana, Kronman, Chanoch, Segall, Yoffi, Velan, Baruch, Shafferman, Avigdor
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.02.2000
• Subjects: Acetylcholinesterase - chemistry; Acetylcholinesterase - metabolism; Cell Line; Chemical Warfare Agents - chemistry; Chemical Warfare Agents - metabolism; Cholinesterase Inhibitors - chemistry; Cholinesterase Inhibitors - metabolism; Humans; Macromolecular Substances; Mass Spectrometry; Models, Molecular; Organophosphates - chemistry; Organophosphates - metabolism; Phosphorylation; Protein Conformation - drug effects
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi992009n

Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed “aging” is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C−O bond, the aging path for adducts with phosphoroamidates (P−N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 ± 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P−N agents can undergo aging through scission of the P−N bond.