Coleman_1993_Mol.Pharmacol_43_217

The interaction of a benzomorphan opiate with the active site of the catalytic subunit of acetylcholinesterase was studied using photoaffinity labeling. UV irradiation of (-)-N-[3H]allylnormetazocine bound to Torpedo acetylcholinesterase resulted in covalent incorporation of 60-70% of the bound ligand. The labeled catalytic subunit was subjected to chemical cleavage with cyanogen bromide and proteolytic degradation with trypsin, chymotrypsin, and staphylococcal V8 protease. The resulting peptide fragments were purified by high performance liquid chromatography and sequenced in the gas phase. The label was not stable under the conditions of the sequencing, but a peptide fragment consisting of Gln74 to Glu82 was reproducibly labeled. These amino acids are located at the rim of a gorge leading to the active site of the enzyme. Molecular modeling studies then demonstrated that these residues can be placed within van der Waals contact of the (-)-N-[3H]allylnormetazocine molecule while it is bound to the active site of the enzyme.