Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenyl-aziridinium.

Abstract

Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N‐dimethyl‐2‐phenyl‐aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270‐278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be prevented by edrophonium; the sequence of one of the peptides assumed to be part of the anionic site in the catalytic centre was found to be DLFR (positions 217‐220). Decamethonium efficiently blocked alkylation by DPA in all three investigated peptides.

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases

“…These structural differences between AChE and BChE and recent investigations employing molecular modeling, site-directed mutagenesis, and studies of the catalytic and inhibitory properties of these mutants lend strong support to the involvement of these aromatic amino acid residues in the binding and selectivity of inhibitors to ChEs. The role of Trp 86(84) in the orientation and stabilization of the quaternary ammonium group of the substrate has been demonstrated by chemical labeling studies (Weise et al, 1990;Kreienkamp et al, 19911, by crystallographic data (Sussman et al, 1991) and site-directed mutagenesis studies for Trp 86(84) in human AChE (Ordentlich et al, 1993). The 2 Phe residues at positions 295(288) and 297(290) define the dimensions of the acyl pocket of mammalian AChEs, markedly reducing BTC hydrolysis and enhancing ATC hydrolysis by forming a clamp around the methyl moiety of the acetoxy group, and restricting its degrees of freedom .…”

Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.

“…Recently we used the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) to localize regions in the primary structure of Torpedo AcChoEase that are involved in the binding of positively charged ligands (25). DPA combines the features of a quaternary ammonium ion with high electrophilic reactivity (26).…”

“…It is an irreversible inhibitor of cholinesterases (27). Two labeled AcChoEase sequences have been identified, one apparently being a component of the peripheral anionic subsite, and the other, of the catalytic-center anionic subsite (25).…”

Novel Anti-Alzheimer's Dimer Bis(7)-Cognitin: Cellular and Molecular Mechanisms of Neuroprotection Through Multiple Targets

“…23 In addition to the catalytic site, AChE possesses another subsite that is able to bind acetylcholine or other quaternary ligands. Using photo-label 24 and affinity label, 25 residues 251 to 264 and residues 270 to 278 in peptide sequences have been identified as part of the peripheral binding sequences. These two peptides lie on the surface of the protein near the rim of the enzyme gorge and bind the bis-quaternary ligands, which usually serve as inhibitors, at one end to the peripheral binding peptide and the other end to the deeper end or the aromatic lining of the enzyme pocket.…”