Exploring the Active Center of Human Acetylcholinesterase with Stereomers of an Organophosphorus Inhibitor with Two Chiral Centers

Abstract

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.

Molecular dynamics study of active-site interactions with tetracoordinate transients in acetylcholinesterase and its mutants

“…(4) The upper pK calculated from the pH rate profile for dealkylation is consistent with that of histidine H + -440. (5) The Glu199Gln mutant of T. californica AChE [16], Glu202Gln mutant of mouse AChE [14], Glu202Gln mutant of human AChE [17,18], Trp86Ala and Phe330Ala mutants of human AChE [19,20] and the corresponding mutations in human butyrylcholinesterase [14,15,21] showed much diminished capacity for dealkylation when inhibited with soman. (6) 99 % of the product of the reaction is derived from a tertiary rather than a secondary carbenium ion [22].…”

“…A major dilemma in postulating the mechanism of dealkylation has been whether or not proton transfer from histisine H + -440 to the OEP occurs to promote dealkylation. Such pre-protonation would be required for the formation of a secondary carbenium ion at Cα in a stepwise mechanism [18][19][20]38]. In contrast, histidine H + -440 stabilizes the developing negative charge on OEP through electrostatic interactions in the concerted mechanism [12][13][14][15], as shown in Scheme 1.…”

Organophosphorus compounds as chemical warfare agents: a review

“…97 In the particular case of soman, there is low selectivity related to the chiral carbon atom. In fact, the P S C S isomer is a bit more toxic than the P S C R , and there is practically no difference between their aging rates, 120 probably because the same carbenium is formed in both cases.…”

Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-agents

“…Therefore, AChE exerts a strong enantioselectivity on chiral OPs, bearing substituents of different sizes. For example, hAChE reacts about 5 ϫ 10 4 times more rapidly with P S diastereoisomers of soman because the large pinacolyl and small methyl substituents fit, respectively, in the choline-binding pocket and acyl-binding pocket (7). The acyl-binding pocket of BChE is much wider than that of AChE and is therefore expected to be less selective for some OPs.…”