LIGAND-INDUCED CONFORMATIONAL-CHANGES IN ACETYLCHOLINESTERASE INVESTIGATED WITH FLUORESCENT PHOSPHONATES

Steady-state kinetic studies have suggested that the catalytic activity of acetylcholinesterase is altered upon binding of certain ligands at a locus removed from the active center. In this study fluorescent phosphonates which conjugate with the active-center serine are employed to demonstrate directly that ligands which associate with the peripheral site alter active-site conformation. The fluorescent reagent [1-(dimethylamino)naphthalene-5-sulfonamido]pentyl methylphosphonofluoridate, when conjugated to the enzyme, proves to be a sensitive probe of active-site conformation. Displacement of propidium, a reversible fluorescent ligand of the peripheral site, was employed to assess peripheral-site occupation by a variety of enzyme inhibitors. Thus, occupation of the peripheral site and induced conformational changes of the active center can be compared directly. The transition-metal ions Zn2+ and Cu2+ are found to be inhibitors of acetylcholinesterase and show an unusually slow onset of inhibition. In the presence of Zn2+ the phosphonate-conjugated enzyme shows an enhanced fluorescence intensity where the emission maximum is shifted to shorter wavelengths; the change in fluorescence intensity occurs slowly, with a rate constant that is comparable to the rate of inhibition of the native enzyme. Displacement of propidium by Zn2+ or d-tubocurarine, a peripheral-site ligand, is found to occur much more rapidly than does the time course of enzyme inhibition or changes in fluorescence of the dansyl phosphonate. Thus, although occupation of the peripheral site is rapid, peripheral-site occupation by certain ligands induces a slow change in active-site conformation. Furthermore, the states induced by the different peripheral-site ligands can be distinguished spectroscopically, indicating nonequivalent conformational effects at the active center upon peripheral-site occupation. © 1979, American Chemical Society. All rights reserved.