"Back door" opening implied by the crystal structure of a carbamoylated acetylcholinesterase

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (ACh E) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholin o)octylcarbamoyleseroline (MF268) is reported at 2.7 Angstrom resolution. I n the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upp er part of the gorge, blocking the entrance of the active site. This preven ts eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not fou nd in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back doo r" opening may occur and shows that the release of products via a "back doo r" is a likely alternative for this enzyme. However, its relevance as far a s the mechanism of substrate hydrolysis is concerned needs to be establishe d. This study suggests that the use of properly designed acylating inhibito rs, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the cl earance of products.