QUATERNARY LIGAND-BINDING TO AROMATIC RESIDUES IN THE ACTIVE-SITE GORGE OF ACETYLCHOLINESTERASE

Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quatern ary ligands were investigated by x-ray crystallography and photoaffini ty labeling. Crystal structures of complexes with ligands were determi ned at 2.8-angstrom resolution. In a complex with edrophonium, the qua ternary nitrogen of the ligand interacts with the indole of Trp-84, an d its m-hydroxyl displays bifurcated hydrogen bonding to two members o f the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp- 84. The bisquatern ary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp -84 and the other to that of Trp-279, near the top of the gorge. The o nly major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium co mplex it lies parallel to the surface of the gorge; in the other two c omplexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labeling by the photo sensitive probe H-3-labeled p-(N,N-dimethylamino)benzenediazonium fluo roborate, which labeled, predominantly, Phe-330 within the active site . Labeling of Trp-279 was also observed. One mole of label is incorpor ated per mole of AcChoEase inactivated, indicating that labeling of Tr p-279 and that of Phe-330 are mutually exclusive. The structural and c hemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary e vidence assigning Trp-84 and Phe-330 to the ''anionic'' subsite of the active site and Trp-279 to the ''peripheral'' anionic site.