Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel

Acetylcholinesterase (EC 3.1.1.7) is a serine hydrolasethat catalyzes the cleavage of acetylcholine. Structuralstudies have revealed that its active site is buried in a20 A˚ deep gorge with a bottleneck [1]. According toa recently developed kinetic model, substrate andproduct molecules follow the same path [2]. A sub-strate molecule first binds to the peripheral site (PAS)at the entrance of the gorge [3] and slides down tothe acylation site (CAS), where it is hydrolyzed andthe products escape the gorge via the entrance. Theactive site gorge is too narrow to allow the crossingbetween a substrate molecule en route to the CASand a product molecule en route to the exit. Conse-quently, at very high substrate concentrations, thereis a traffic jam preventing the exit of the reactionproduct through the main entrance, resulting in inhi-bition [4].However, molecular dynamics experiments have pro-vided evidence for a loop movement leading to the for-mation of a back door suitable for product exit [5].Locking the loop with salt or disulfide bridges [6,7]had no significant effect on the kinetics parameters,indicating that exit through the back door is not themain exit route for the product. However, residualactivity upon fasciculin binding suggests that the backdoor route might become the most important routewhen the main entrance is blocked [8,9]. Recent kineticcrystallography studies provide some structural insightsregarding the putative backdoor. Conformationchanges of Trp84, which belongs to the backdoorregion of Torpedo californica acetylcholinesterase, sug-gest that this residue might behave like a revolvingdoor [10]. In addition, Nachon et al. [11] reported thatthe back door region of the Drosophila acetylcholines-