title: Structural insights into substrate traffic and inhibition in acetylcholinesterase.
abstract: Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.
authors.affiliation: Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale (CEA/CNRS/UJF), Grenoble Cedex, France.
authors.forename: Jacques-Philippe
authors.initials: JP
authors.lastname: Colletier
authors.forename: Didier
authors.initials: D
authors.lastname: Fournier
authors.forename: Harry M
authors.initials: HM
authors.lastname: Greenblatt
authors.forename: Jure
authors.initials: J
authors.lastname: Stojan
authors.forename: Joel L
authors.initials: JL
authors.lastname: Sussman
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Zaccai
authors.forename: Israel
authors.initials: I
authors.lastname: Silman
authors.forename: Martin
authors.initials: M
authors.lastname: Weik
mesh_terms: D000109:Acetylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism*; D000122:Acetylthiocholine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002800:Cholinesterase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity; D014101:Torpedo
chemical_list: D002800:Cholinesterase Inhibitors; C034175:trimethyl(4-oxopentyl)ammonium; D000122:Acetylthiocholine; D000110:Acetylcholinesterase; D000109:Acetylcholine