Acetylcholinesterase inhibition via a series of polyacene probe molecules: an example of integrated probe management techniques.

Abstract

Abstract Rate control in acetylcholinesterase (AChE) appears to involve a single anionic site whose anionic center binding controls rate-related changes in the free (E) and acyl (EA) enzyme forms. Changes in enzyme conformation associated with ligand binding at this center affect AChE's acylation and hydrolysis rates. This comprehensive study of probe ligand binding to AChE includes data based on both steady-state kinetic inhibition and ligand fluorescence quenching data. It was made possible via the custom synthesis of numerous probe molecules of known stereochemistry. These probes act as rulers of the E and EA enzyme forms of AChE and provide comparative data from both kinetic and thermodynamic (fluorescence quenching) based binding constants. Data obtained from the binding of these probes indicate that AChE's anionic center in the E conformer lies at the bottom of a sterically restricted narrow cleft in the protein surface. This is consistent with X-ray crystallographic data for AChE. The anionic center of the EA conformer, responsible for allosteric rate control, has an altered stereochemistry. It is characterized by a decrease in the relative affinity for hydrophobic cations and by an ability to bind large organic cations. Binding of substrate, H + , or organic cations at this anionic center controls k 2 in the E conformer and k 3 in the EA conformer. Substrate binding to the E conformer results in the ES enzyme form which maximizes k 2 . Binding studies based on fluorescence quenching of appropriate acridine ligands with both the E and EA enzyme form indicate binding at only a single anionic site. No ternary complex (EII' or EAII') resulting from two site binding to separate active and allosteric sites was detected. In AChE, nonspecific, low level affinities at surface anionic or hydrophobic areas is ubiquitous; however, it is not great enough to be responsible for rate controlling activities. These low affinity casual bindings do not appear to modify the fundamental activity of either the E or EA conformers and thus have little effect on enzyme rate control.