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.