Abstract

The synthesis of 4-azido-2-nitrobenzyltrimethylammonium fluoroborate, a photoaffinity label for cholinergic ligand binding sites, is described. The effect of this compound on acetylcholinesterase from Torpedo californica electric tissue has been investigated. In the dark it inhibits the enzyme reversibly, with a mixed competitive-noncompetitive mechanism (K_i = 7 µM). After irradiation at a wavelength of about 350 nm, the compound binds irreversibly and highly specifically to anionic binding sites of the enzyme. This was shown by experiments with the radioactive label and with cholinergic ligands containing quaternary ammonium groups, which protect the enzyme against inactivation and incorporation of the label. Experiments with Tetram and its triethyl derivative indicate that mainly the anionic subsite within the active site is labeled. After protection of the active site with edrophonium or carbamoylcholine, a peripheral anionic site can be blocked and labeled selectively with the photoaffinity label. This labeling of the peripheral site alters the reversible inhibition by the photolabel in the dark from a mixed competitive-noncompetitive mechanism to a purely competitive pattern (K_i = 16 µM for competition of the photoaffinity label in the dark with the substrate acetylthiocholine).