Unique Push−Pull Mechanism of Dealkylation in Soman-Inhibited Cholinesterases

Publisher Website
Google Scholar
Abstract
The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) and fetal bovine serum (FBS) acetylcholinesterase (AChE) inactivated with P(−)C(+) and P(−)C(−) 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 4.0 ± 0.2 °C. The rate constant versus pH profiles were fit to a bell-shaped curve for all adducts. Best fit parameters are pK1 4.4−4.6 and pK2 6.3−6.5 for Ee AChE and pK1 4.8−5.0 and pK2 5.8 for FBS AChE. The pKs are consistent with catalytic participation of the Glu199 anion and HisH+440. Maximal rate constants (kmax) are 13−16 × 10-3 s-1 for Ee AChE and 8 × 10-3 s-1 for FBS AChE. The solvent isotope effects at the pH maxima are 1.1−1.3, indicating unlikely proton transfer at the enzymic transition states for the dealkylation reaction. Slopes of log rate constant versus pH plots are near 1 at 25.0 ± 0.2 °C between pH 7.0 and 10.0. In stark contrast, the corresponding adducts of trypsin are very stable even at 37.0 ± 0.2 °C. The rate constants for diastereomers of soman-inhibited trypsin at 37.0 ± 0.2 °C are pH independent and ∼104 times smaller than kmax for analogous adducts with AChE. Dealkylation in soman-inhibited AChEs is estimated to occur at >1010 times faster than a plausible nonenzymic reaction. Up to 40% of the catalytic acceleration can be attributed to an electrostatic push, and an electrostatic pull provides much of the balance. The results of this work together with results of a product analysis by Michel et al. (1969) can be explained by an initial and rate-determining methyl migration from Cβ to Cα. This is driven by the high electron density of residues (Glu199 and Trp84) at a crowded active site and may be concerted with C−O bond breaking. The positive charge at the rate-determining transition state is distributed between Cβ and His440. A tertiary carbocation may have a fleeting existence before it is trapped by water or neighboring electrons which is likely to be promoted by Glu199 as the proton acceptor.