PARALLEL MECHANISMS IN ACETYLCHOLINESTERASE-CATALYZED HYDROLYSIS OF CHOLINE ESTERS

The mechanisms of acetylcholinesterase (AChE)-catalyzed hydrolyses of four choline esters, (acetylthio)-choline, (propanoylthio)choline, (bu tanoylthio)choline, and benzoylcholine, have been compared by measurin g relative activities and pH-rate effects. The reactivity of Electroph orus electricus AChE toward these substrates decreases in the above or der and spans a 1500-fold range of k(cat) and a 2300-fold range of k(c at)/K(m). The pH-V/K profile for (acetylthio)choline hydrolysis shows that activity depends on the basic form of an active site residue, pro bably H440, that has a pK(a) = 6.22 +/- 0.03 (Rao, M.; et al. J. Am. C hem. Soc. 1993, 115, in press; ref 23). The pH-V/K profile for (propan oylthio)choline shows similar behavior and gives a pK(a) = 6.42 +/- 0. 03. The pH-V/K profile for (butanoylthio)-choline hydrolysis, on the o ther hand, shows that activity depends on the basic forms of three act ive 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 substra te, benzoylcholine, pH-V and pH-V/K profiles depend only on the ioniza tions 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 benzo ylcholine hydrolysis are (D)V/K = 1.91 +/- 0.06 and (D)V = 1.7 +/- 0.3 , and proton inventories of these parameters suggest that a carboxylat e residue functions as a general base catalyst. Torpedo californica AC hE also displays a high kinetic discrimination for (acetylthio)choline versus (butanoylthio)choline; the ratio of k(cat) values for the resp ective substrates is 3800, while the corresponding k(cat)/K(m) ratio i s 850. The pH-V/K profile for hydrolysis of (acetylthio)choline depend s on a single residue that has pK(a) = 6.33 +/- 0.03, while that for h ydrolysis of (butanoylthio)choline has a maximum at pH approximately 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 ace tylcholinesterase-catalyzed hydrolysis of choline esters. The biomimet ic substrate (acetylthio)choline and the homologue (propanoylthio)chol ine are hydrolyzed via nucleophilic and general acid-base catalysis by the active site triad S200-H440-E327. As the substrate reactivity dec reases, the mechanism shifts progressively from triad catalysis to one that likely involves general base catalysis by E199 of direct water a ttack on the scissile carbonyl. Molecular modeling suggests that the s terically circumscribed acyl-binding site accommodates the acetyl and propanoyl functions of choline substrates. However, the sterically enc umbered butanoyl and benzoyl functions are less well accommodated in t he 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.