title: The architecture of human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors.
abstract: The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
authors.affiliation: Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona.
authors.forename: A
authors.initials: A
authors.lastname: Ordentlich
authors.forename: D
authors.initials: D
authors.lastname: Barak
authors.forename: C
authors.initials: C
authors.lastname: Kronman
authors.forename: N
authors.initials: N
authors.lastname: Ariel
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; D001665:Binding Sites; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D010766:Phosphorylation; D013329:Structure-Activity Relationship
chemical_list: D002800:Cholinesterase Inhibitors; D009943:Organophosphorus Compounds; D000110:Acetylcholinesterase