SIMPLE GENERAL ACID-BASE CATALYSIS OF PHYSIOLOGICAL ACETYLCHOLINESTERASE REACTIONS

Elements of transition-state stabilization by proton bridging have been characterized by measuring solvent isotope effects and proton inventories for hydrolyses of (acetylthio)choline (ATCh), (propionylthio)choline (PrTCh), and (butyryl-thio)choline (BuTCh) catalyzed by acetylcholinesterases (AChEs) from Electrophorus electricus, fetal bovine serum, human erythrocytes, and Torpedo californica. For the Electrophorus enzyme, the acylation rate constant, k(cat)/K(m) { = (V/K)/[E]T}, decreases in the order ATCh > PrTCh >> BuTCh. Solvent isotope effects for V/K of ATCh hydrolysis are usually within experimental error of unity, which is consistent with rate determination of the acylation stage of catalysis by a physical step, such as substrate diffusion. However, as substrate reactivity decreases the isotope effect increases, which indicates that the transition state of a chemical step is increasingly rate determining. A linear proton inventory for V/K of BuTCh hydrolysis indicates that this chemical transition state is stabilized by single proton transfer. Solvent isotope effects for V are approximately 2, and the corresponding proton inventories are invariably linear, irrespective of the source of AChE, the choice of substrate, the ionic strength of the medium, or the presence of the detergent TX100. The consistency of the results strongly suggests that AChE stabilizes chemical transition states of physiological reactions by one-proton, simple general acid-base catalysis. Therefore, elaborate themes in transition-state stabilization by proton transfer, such as multifunctional or charge-relay catalyses, do not appear to operate in the physiological functioning of AChE.