The architecture of human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors

J Biol Chem. 1996 May 17;271(20):11953-62. doi: 10.1074/jbc.271.20.11953.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetylcholinesterase / chemistry*
  • Acetylcholinesterase / metabolism
  • Binding Sites
  • Cholinesterase Inhibitors / pharmacology*
  • Humans
  • Hydrogen Bonding
  • Kinetics
  • Mutagenesis, Site-Directed
  • Organophosphorus Compounds / pharmacology*
  • Phosphorylation
  • Structure-Activity Relationship


  • Cholinesterase Inhibitors
  • Organophosphorus Compounds
  • Acetylcholinesterase