The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration
on the aging process were examined for human acetylcholinesterase (HuAChE) and its
selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl
methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman
diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers.
Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than
that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray
mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited
very limited sensitivity to the chirality of the alkoxy group Calpha of either PS-
or PR-diastereomers. These stereoselectivities presumably reflect the relative participation
of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the
respective covalent conjugates, and therefore could be utilized for further probing
of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes
carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate
that stereoselectivity with respect to the soman phosphorus chirality depends on the
structure of this binding subsite, but this stereoselectivity cannot be explained
only by limitation in the capacity to accommodate the PR-diastereomers. In addition,
these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman
over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme
W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions
in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket
(F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS-
over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant
enzymes tested practically lacked stereoselectivity with respect to the Calpha of
the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem
to affect the rate-determining step of the dealkylation process, possibly because
both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.