Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers

The stereoselectivity of the phosphonylation reaction and the effects of ad duct configuration on the aging process were examined for human acetylcholi nesterase (HuAChE) and its selected active center mutants, using the four s tereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The r eactivity of wild type HuAChE toward the P-S-soman diastereomers was 4.0-7. 5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the P SCS-somanyl-HuAChE conjugate was also >1.6 x 104-fold faster than that of t he corresponding PRCS-somanyl adduct, as shown by both reactivation and ele ctrospray mass spectrometry (ESI/MS) experiments. On the other hand, both p rocesses exhibited very limited sensitivity to the chirality of the alkoxy group C-alpha of either P-S- or P-R-diastereomers. These stereoselectivitie s presumably reflect the relative participation of the enzyme in stabilizat ion of the Michaelis complexes and in dealkylation of the respective covale nt conjugates, and therefore could be utilized for further probing of the H uAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) i ndicate that stereoselectivity with respect to the soman phosphorus chirali ty depends on the structure of this binding subsite, but this stereoselecti vity cannot be explained only by limitation in the capacity to accommodate the P-R-diastereomers. In addition, these acyl pocket enzyme mutants displa y some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, w hile reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRC S-soman resembles that of the wild type HuAChE. Residue substitutions in th e H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocke t (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the P-S-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the C-alpha of the alkoxy moiety. Thus, the inherent asymme try 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-s omanyl moieties yield the same carbocationic intermediate.