Parallel mechanisms in acetylcholinesterase-catalyzed hydrolysis of choline esters

Article abstract of DOI:10.1021/ja00076a002

The mechanisms of acetylcholinesterase (AChE)-catalyzed hydrolyses of four choline esters, (acetylthio)-choline, (propanoylthio)choline, (butanoylthio)choline, and benzoylcholine, have been compared by measuring relative activities and pH-rate effects. The reactivity of Electrophorus electricus AChE toward these substrates decreases in the above order and spans a 1500-fold range of k_cat and a 2300-fold range of k_cat/K_m. The pH-V/K profile for (acetylthio)choline hydrolysis shows that activity depends on the basic form of an active site residue, probably H440, that has a pK_a = 6.22 ± 0.03 (Rao, M.; et al. J. Am. Chem. Soc. 1993, 115, in press; ref 23). The pH-V/K profile for (propanoylthio)choline shows similar behavior and gives a pK_a = 6.42 ± 0.03. The pH-V/K profile for (butanoylthio)-choline hydrolysis, on the other hand, shows that activity depends on the basic forms of three active site residues, two that have pK_a values of 4.72 ± 0.02 and one that has a pK_a value of 6.3 ± 0.1. For the least reactive substrate, benzoylcholine, pH-V and pH-V/K profiles depend only on the ionizations of the residues that have the lower pK_a values, 4.77 ± 0.03 and 4.71 ± 0.03, respectively. The solvent isotope effects for benzoylcholine hydrolysis are _DV/K = 1.91 ± 0.06 and ^DV = 1.7 ± 0.3, and proton inventories of these parameters suggest that a carboxylate residue functions as a general base catalyst. Torpedo californica AChE also displays a high kinetic discrimination for (acetylthio)-choline versus (butanoylthio)choline; the ratio of k_cat values for the respective substrates is 3800, while the corresponding k_cat/K_m ratio is 850. The pH-V/K profile for hydrolysis of (acetylthio)choline depends on a single residue that has pK_a = 6.33 ± 0.03, while that for hydrolysis of (butanoylthio)choline has a maximum at pH ～5.5 and depends on amino acid residues that have pK_a values of 4.99 ± 0.07 and 6.1 ± 0.3. Therefore, parallel mechanisms operate in acetylcholinesterase-catalyzed hydrolysis of choline esters. The biomimetic substrate (acetylthio)choline and the homologue (propanoylthio)choline are hydrolyzed via nucleophilic and general acid-base catalysis by the active site triad S200-H440-E327. As the substrate reactivity decreases, the mechanism shifts progressively from triad catalysis to one that likely involves general base catalysis by E199 of direct water attack on the scissile carbonyl. Molecular modeling suggests that the sterically circumscribed acyl-binding site accommodates the acetyl and propanoyl functions of choline substrates. However, the sterically encumbered butanoyl and benzoyl functions are less well accommodated in the acyl-binding site, and thus the corresponding substrates apparently occupy an alternate binding site in the active site gorge from which catalysis by E199 is effected.