Acetylcholinesterase (AChE) is an enzyme whose role in human physiology and health is well documented. Besides its role in cholinergic neurotransmission, AChE is an important pharmacological and toxicological target. Reversible inhibitors of the enzyme are used to treat glaucoma and myasthenia gravis, and recently to allay the symptoms of Alzheimer's disease. Some irreversible inhibitors are among the most toxic substances ever synthesized and form the basis of chemical warfare plans. Despite the importance of AChE catalysis, little is known of the chemical events by which it achieves its catalytic aim. This is likely due to the fact that AChE is such a powerful catalyst that nonchemical steps appear to control the reaction rate. For the natural substrate acetylcholine the rate-determining step monitored by kcat/Km is likely diffusion of the substrate into the active site, whereas for good acetate ester substrates a rate determining induced fit accompanying substrate binding has been suggested. The primary objective of this proposal is to develop amide and anilide substrates of AChE that are close isosteres of good ester substrates. It is anticipated that the lower reactivity of amides and anilides will expose chemical catalytic events as rate determining for kcat/Km, which always monitors steps in the acylation manifold of the AChE acylenzyme mechanism. A range of experimental probes will be used to determine structural features of transition states of AChE-catalyzed hydrolyses of anilides and amides: 1) Solvent deuterium isotope effects and pH-rate effects will be measured to assess the role of proton transfer. 2) Substrate secondary deuterium isotope effects will be measured to detect bond making/breaking processes involving heavy (i.e., nonhydrogen) atoms. 3) The rate-determining step monitored by kcat measurements will be determined by using ester and anilide substrates that have identical acyl structures and, if need be, by trapping radiolabelled acylenzymes. 4) Modulation of transition state structure on interaction of the enzyme with activity-modifying ligands or with lipid surfaces will be probed. In vivo, AChE is an extrinsic membrane-bound enzyme, and (in vitro) AChE activity is sensitive to the physical state of the membrane. Therefore, the long-term aim of the research described herein is to determine the functional significance of AChE-membrane interactions (as a model for lipid-protein interactions) by assessing the effects of changes in lipid dynamics on catalytic transition state structures.
