Abstract

Organophosphate adducts of serine esterase can undergo a dealkylation reaction which converts phosphylated enzymes into “aged” species which can not be reactivated. The molecular basis for the resistance to reactivation of aged enzymes is still unclear. In the present work, we focused on the importance of water in the mechanism of dealkylation. For this, we investigated the effects of hydrostatic and osmotic pressures on the rate of aging of diisopropyl-phosphorylated wild-type and mutants of human butyrylcholinesterase (DIP-wtBuChE, E197D and D70G mutants). Hydrostatic pressure strongly increased the rate of aging of wt-BuChE but had little effect on the aging of DIP-mutants. Unlike hydrostatic pressure, osmotic pressure decreased the rate of aging of both wild-type and mutant enzymes, giving large positive Δνπ ≠. Our results showed that water is needed for stabilization of the transition state and that residues E197 and D70 control the water/H bond network in the active site gorge.

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