ACETYLCHOLINESTERASE PERIPHERAL ANIONIC SITE DEGENERACY CONFERRED BY AMINO-ACID ARRAYS SHARING A COMMON CORE

Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combinati on of kinetic studies with 19 single and multiple HuAChE mutants, fluo rescence binding studies with the Trp-286 mutant, and by molecular mod elling. Mutants were analyzed with three structurally distinct positiv ely charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-di methylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutati ons of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulte d in up to 10-fold increase in inhibition constants for PAS ligands, w hereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conf ormation of both the active site and the PAS (Shafferman, A., Velan, B ., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y ., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) a s demonstrated by the several hundred-fold increase in K-i for D74N in hibition by the bisquaternary ligands decamethonium and BW284C51. Base d on these studies, singular molecular models for the various HuAChE i nhibitor complexes were defined. Yet, for the decamethonium complex tw o distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp -286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This f unctional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-c ation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.