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

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Author: Ordentlich, A. · Barak, D. · Kronman, C. · Benschop, H.P. · Jong, L.P.A. de · Ariel, N. · Barak, R. · Segall, Y. · Velan, B. · Shafferman, A.
Type:article
Date:1999
Source:Biochemistry, 10, 38, 3055-3066
Identifier: 234974
doi: doi:10.1021/bi982261f
Keywords: Cholinesterase reactivator · Soman · Binding site · Chirality · Dealkylation · Enzyme activity · Enzyme analysis · Enzyme binding · Enzyme specificity · Enzyme structure · Enzyme substrate · Human cell · Protein expression · Stereochemistry · Stereoisomerism · Alkylation · Amino acid substitution · Binding sites · Cell line · Cholinesterase inhibitors · Humans · Hydrogen Bonding · Kidney · Macromolecular substances · Models, Molecular · Mutagenesis, Site-Directed · Stereoisomerism · Acetylcholinesterase, EC 3.1.1.7 · Alanine, 56-41-7 · Phenylalanine, 63-91-2 · Phosphorus, 7723-14-0 · Soman, 96-64-0

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 P(S)-soman diastereomers was 4.0-7.5 x 104-fold higher than that toward the P(R)- diastereomers. Aging of the P(S)C(S)-somanyl-HuAChE conjugate was also > 1.6 x 104-fold faster than that of the corresponding P(R)C(S)-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 C(α) of either P(S)- or P(R)-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 P(R)-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the P(R)C(R)-soman over the P(R)C(S)-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the P(R)C(S)- 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 P(S)C(S)- over the P(S)C(R)-stereomer. Aging of the P(S)-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the C(α) 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 P(S)C(S)- and the P(S)C(R)-somanyl moieties yield the same carbocationic intermediate.