tailieunhanh - Báo cáo khoa học: Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel

To test a product exit differing from the substrate entrance in the active site of acetylcholinesterase (EC ), we enlarged a channel located at the bottom of the active site gorge in the Drosophilaenzyme. Mutation of Trp83 to Ala or Glu widens the channel from 5 A˚ to 9 A ˚ . | ỊFEBS Journal Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel Florian Nachon1 Jure Stojan2 and Didier Fournier3 1 Departement de Toxicologie CRSSA Grenoble France 2 Institute of Biochemistry MedicalFaculty Ljubljana Slovenia 3 IPBS Universite PaulSabatier CNRS Toulouse France Keywords acetycholinesterase back door inhibition substrate traffic Correspondence F. Nachon Unite d enzymologie Departement de Toxicologie Centre de Recherches du Service de Sante des Armees CRSSA 24 Avenue des Maquis du Gresivaudan 38700 La Tronche France Fax 33 476636962 Tel 33 476639765 E-mail fnachon@ Received 18 December 2007 revised 14 March 2008 accepted 18 March 2008 To test a product exit differing from the substrate entrance in the active site of acetylcholinesterase EC we enlarged a channel located at the bottom of the active site gorge in the Drosophila enzyme. Mutation of Trp83 to Ala or Glu widens the channel from 5 A to 9 A. The kinetics of substrate hydrolysis and the effect of ligands that close the main entrance suggest that the mutations facilitate both product exit and substrate entrance. Thus in the wild-type the channel is so narrow that the back door is used by at most 5 of the traffic with the majority of traffic passing through the main entrance. In mutants Trp83Ala and Trp83Glu ligands that close the main entrance do not inhibit substrate hydrolysis because the traffic can pass via an alternative route presumably the enlarged back channel. doi Acetylcholinesterase EC is a serine hydrolase that catalyzes the cleavage of acetylcholine. Structural studies have revealed that its active site is buried in a 20 A deep gorge with a bottleneck 1 . According to a recently developed kinetic model substrate and product molecules follow the same path 2 . A substrate molecule first binds to the peripheral site PAS at the entrance .