Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase

Acetylcholinesterases (AChEs) form conjugates with certain highly toxic org anophosphorus (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 phosp honates 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 t he 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 tha t of the wild-type enzyme. This is in marked contrast to the large effect o f these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be invol ved. 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 HuBChE mutants (E202Q, F338A, F29 5A, 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 decreas e of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass de crease 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 th e P-N bond.