CONFORMERS OF ACETYLCHOLINESTERASE - A MECHANISM OF ALLOSTERIC CONTROL

Rate control in acetylcholinesterase (AChE) involves a single anionic site whose anionic center controls rate-related biochemical and confor mational changes in the E (free enzyme) and EA (acylated enzyme) confo rmers. Change in conformer structure and biochemistry affect binding, acylation, and hydrolysis. It is significant that the anionic-esterati c intersite distance is not altered during conformer change as E is co nverted to EA. In this enzyme system, cationic acetylcholine and anion ic AChE are true structural, functional, and biochemical counterparts. The anionic center in the E conformer lies at the bottom of a sterica lly restricted, hydrophobic cleft <8 Angstrom wide at the top and >3 A ngstrom wide at the bottom, while the anionic center in the EA conform er is relatively open. It is characterized by a decrease in the relati ve binding of hydrophobic cations and by an ability to bind large orga nic cations. Binding of acetylcholine, H+, or organic cations at the a nionic site controls k(2(acylation)) in the E conformer and k(3(hydrol ysis)) in the EA conformer. Acetylcholine binding forms the ES complex in which the cation maximizes k(2). In the EAS complex, the cation re duces k(3) and provides allosteric control. Anionic site structure and biochemistry and the effect of pH on k(2) and k(3) differentiates ACh E from butyrlcholinesterase. This comprehensive study of kinetic and t hermodynamic processes in AChE was made possible by the synthesis and/ or use of families of over 30 cationic and acylation probes of known s tereochemistry. They act as rulers of the E and EA conformers of AChE and provide comparative data on kinetic-based and thermodynamic-based constants. Cationic inhibitors affect decarbamylation rates in AChE an d provide an additional set of comparative data related to the mechani sm of substrate hydrolysis by AChE. Acridine araphanes are unique neur al receptor and cholinergic enzyme probes. Their parallel plane and co planar conformations are related to bridge length. Two parallel plane acridine araphanes are pure uncompetitive inhibitors of AChE. Scatchar d plots of the binding of methylacridinium and 9-aminoacridine with th e E conformer and 9-aminoacridine with the EA conformer indicate bindi ng at a single anionic site. No ternary complex (EII or EAII) from two -site binding was detected. In AChE, nonspecific, low-level binding at surface ionic and hydrophobic areas is ubiquitous. Binding affinity d ifferences greater than two orders of magnitude distinguish binding at the anionic site from low level binding at surface moieties. Surface binding provides environmental and stability changes in the enzyme but does not modify the fundamental biochemistry of the E and conformers.