Acetylcholinesterase (AChE) is an enzyme with rich biomedical, pharmacological and toxicological history. This is undoubtedly due to the central role tat AChE plays, along with the ACh receptor, in cholinergic neurotransmission. AChE is the physiological target of chemical warfare agents such as sarin and soman, which are among the most toxic substances ever invented by man. Other AChE inhibitors are useful in the treatment of myasthenia gravis, glaucoma, and perhaps Alzheimer's disease. Still other species specific AChe inhibitors are useful pest control agents. Given the obvious financial commitment that American society has in AChE, it is surprising that much is yet to be discovered about the chemical events of AChE catalysis. During the past two years of support from NS21334, the applicant's research program has begun the characterization of the anatomy of AChE catalysis (i.e. reaction dynamics and thermodynamics, rate-limiting steps, chemical transition state structures). This work has shown that AChE reactions are usually rate limited by virtual transition states that are comprised of contributions from partially rate limiting physical and chemical transition states. This application for continuation of NS21334 seeks support to continue these investigations of the anatomy of AChE catalysis, and to explore the possibility that AChE and butyrylcholinesterase (BuChE) are mechanistically related. An array of probes will be used to study reaction dynamics and virtual transition states for BuChE and (especially) AChE catalyzed hydrolyses of anilides, aryl esters and thiocholine esters. These include: a) pL(L=H,D)- Rate profiles; b) solvent isotope effects and proton inventories. These probes are designed to assess the contribution to rate determination of and structural features of transition state that are stabilized by proton bridging. c) Substrate isotope effects. Heavy atom and secondary isotope effects will be used to characterize the core structure (i.e. substrate-derived portion) of AChE and BuChE catalytic transition states. d) Temperature effects; e) Effects of activity-modifying ligands. These probes will be applied to manipulate the relative rate determination by physical and chemical transition states of AChE reactions, thereby hopefully exposing chemical transition states to structural characterization. It is anticipated that the investigations outlined in this proposal will result in a delineation of AChE reaction dynamics and chemical transition state structures that is rivaled by few other hydrolytic enzyme mechanisms.
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