PH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants

Abstract

The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) acetylcholinesterase (AChE), fetal bovine serum (FBS) AChE (1), mouse (Mo) AChE and human (Hu) butyrylcholinesterase (BChE) inactivated with P(S)C(S) and P(S)C(R) 2-(3, 3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 25.0 ± 0.1 EC with Mo AChE and at 4.0 ± 0.1 EC with the other cholinesterases.(2) Analogous studies were carried out with the E202Q mutant of Mo AChE and the E197Q, E197D, E197G and W82A mutants of Hu BChE at 25.0 ± 0.1 EC (2). Best fit parameters for the asymmetric bell-shaped curves observed for the adducts of Ee, FBS and Mo AChE are pK1 = pK2 = 4–4.9 and pK3 = 5.2–6.6. These pKs are consistent with the participation of two carboxylate ions, possibly from E327 and E199, and His440H+ in catalysis of the dealkylation reaction by AChEs. The E199Q mutant of Mo AChE inactivated with the soman diastereomers yielded a single pK with values of 5.5–5.8. Nearly symmetric pH curves for soman-inhibited Hu BChE and its E197D mutant gave pK2 of 3.7–4.6 and pK2 of 7.3–8, but for the corresponding adducts of the E199Q mutant pK2 is a low 5. The dealkylation in soman-inhibited Hu BChE is then consistent with participation of one carboxylate ion side chain and His447H+. Maximal rate constants (kmax) are 1–6 min−1 for AChEs and 2 min−1 for Hu BChE at 25.0 EC. The solvent isotope effects at the pH maxima are 1.1–1.3 indicating unlikely proton transfer or preprotonation at the enzymic transition states for the dealkylation reaction.