title: Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase.
abstract: 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.
authors.affiliation: Departments of Organic Chemistry, Biochemistry and Molecular Biology, and Analytical Chemistry, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Barak
authors.forename: A
authors.initials: A
authors.lastname: Ordentlich
authors.forename: D
authors.initials: D
authors.lastname: Kaplan
authors.forename: R
authors.initials: R
authors.lastname: Barak
authors.forename: D
authors.initials: D
authors.lastname: Mizrahi
authors.forename: C
authors.initials: C
authors.lastname: Kronman
authors.forename: Y
authors.initials: Y
authors.lastname: Segall
authors.forename: B
authors.initials: B
authors.lastname: Velan
authors.forename: A
authors.initials: A
authors.lastname: Shafferman
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002619:Chemical Warfare Agents / Q000737:chemistry* / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008958:Models, Molecular; D010755:Organophosphates / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects
chemical_list: D002619:Chemical Warfare Agents; D002800:Cholinesterase Inhibitors; D046911:Macromolecular Substances; D010755:Organophosphates; D000110:Acetylcholinesterase; C009374:tabun