“Back Door” Opening Implied by the Crystal Structure of a Carbamoylated Acetylcholinesterase

Abstract

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline ( MF268) is reported at 2.7 Å resolution. In 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 upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

The Kinetics of Inhibition of Human Acetylcholinesterase and Butyrylcholinesterase by Two Series of Novel Carbamates

“…In rhAChE, larger N-alkyl groups may be accommodated in a region that spans the less crowded entrance to the gorge, thereby providing tighter binding with less steric constraint. This is consistent with the TcAChE crystal structures for mono N-substituted carbamates with a bulky N-substituent (Bartolucci et al, 1999(Bartolucci et al, , 2006.…”

“…Despite these unexplained kinetic discrepancies between the two enzymes, the crystal structures of covalent conjugates, formed between TcAChE and bulky N-substituted mono-and dialkyl-carbamates with large leaving groups, provide information on the relative orientation of the covalently bound carbamyl moiety and may assist in the rationalization of results obtained from the kinetics of inhibition of rhAChE by the AI and PE series. While the N-methyl,Nethyl carbamyl adduct of rivastigmine disrupted the catalytic triad (Bar-On et al, 2002), the bulky mono-N-alkyl carbamates of (N-cis-2,6-dimethylmorpholino-octyl chain (Bartolucci et al, 1999) and the (N-2Ј-ethyl phenyl) moiety of ganstigmine (Bartolucci et al, 2006) did not change the orientation of His440 of the catalytic triad of TcAChE. In the latter cases, resistance of the carbamylated enzyme to spontaneous reactivation was rationalized by the existence of a strong hydrogen bond between the NH of the mono N-carbamyl moiety and the N 2 atom of the catalytic His440 that is required for catalysis.…”

Axial Conformation of 3-Methyl-2-butenoyl Group in Pyranocoumarin Ring Endows Biological Activity of (+)-Decursin

“…The molecule in red is MF268 inhibitor. 24 The protein structure was obtained from RCSB protein databank (1OCE) and processed by Swiss-PdbViewer 3.7.…”

Cholinesterases: Structure, Role, and Inhibition

“…Ovom je metodom kod AChE predviđeno i postojanje alternativnog izlaza (engl. back door hypothesis) kojim bi se kolin nastao pri hidrolizi supstrata mogao eliminirati iz aktivnog mjesta (107,108). Specifi čan oblik aktivnog mjesta i iznimno brza hidroliza fiziološkog supstrata acetilkolina podupiru ovu hipotezu, ali još nedostaje eksperimentalni dokaz (107,108).…”