KINETICS OF ASSOCIATION BETWEEN BISQUATERNARY AMMONIUM LIGANDS AND ACETYLCHOLINESTERASE - EVIDENCE FOR 2 CONFORMATIONAL STATES OF THE ENZYME FROM STOPPED-FLOW MEASUREMENTS OF FLUORESCENCE

Bisquaternary ammonium ligands in which the quaternary groups are separated by 14 .ANG. are known to bind with high affinity to acetylcholinesterase. A series of bisquaternary ligands containing a benzoquinone moiety exhibit absorption spectra which overlap the fluorescence emission of the tryptophanyl residues on the protein. Upon complexation with actylcholinesterase, quenching of protein fluorescence occurs along with a shift of emission maximum to shorter wavelength. The latter phenomenon suggests that a conformational change is associated with ligand binding. A study of the kinetics of complexation using stopped-flow instrumentation reveals that a rapid bimolecular step (k = 9.7 .times. 107 M-1s-1) is followed by a relatively slow unimolecular (k = 44 s-1) interconversion of enzyme species. The unimolecular step, which appears to involve an isomerization of 2 enzyme forms that exist prior to ligand binding, occurred for all the bisquaternary ligands examined. The kinetics of complexation, where a bimolecular rate below diffusion-controlled rates and a slow isomerization of the enzyme are observed, appear to be unique to the bisquaternary compounds since ligands which bind exclusively to the active center or peripheral anionic site exhibit association kinetics approximating that of a diffusion-controlled reaction. The change in conformation may well be associated with the necessity of obtaining the proper spatial arrangement between the 2 anionic sites on acetylcholinesterase at which the quaternary groups bind.