Equilibrium and kinetic studies of the interaction of site-specific ligands with acetylcholinesterase from Electrophorus electricus

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Abstract
The interactions of edrophonium chloride, gallamine triiodide and propidium diiodide with affinity-purified acetylcholinesterase from E. electricus were examined under conditions of low ionic strength (0.001 M Tris, pH 8.0) using kinetic and fluorescence titration techniques. Edrophonium is a competitive inhibitor of the steady-state hydrolysis of acetylthiocholine, with an inhibition constant, Kcomp, of 1.2 .times. 10-8 M. Double reciprocal plots in the presence of either gallamine or propidium are nonlinear. Similarly, the pre-steady-state carbamoylation of the enzyme by 7-(dimethylcarbamoyloxy)-N-methyl quinolinium iodide is competitively inhibited by edrophonium, whereas the intercepts of the double reciprocal plots of pseudo-first-order rate constant of carbamoylation vs. substrate concentration are displaced downwards in the presence of gallamine or propidium. These results, and those of equilibrium binding studies utilizing the fluorescence properties of bound propidium, suggest that gallamine and propidium compete for a peripheral class of anionic sites on the enzyme, whereas edrophonium binds to the anionic subsite of the catalytic site. The characteristics of propidium binding to the eel enzyme differ from those previously observed with enzyme isolated from Torpedo californica. Whereas the tetrameric Torpedo enzyme possesses 4 binding sites of equal affinity for propidium, the eel enzyme appears to have 2 classes of propidium binding site. One set of approximately 2 sites/tetramer is characterized by a Kd of approximately 2-5 .times. 10-8 M; a 2nd set of 2 sites bind propidium with a Kd of 4 .times. 10-6 M. Possible reasons for these differences are discussed.