Abstract

Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate.

Highlights

  • Organophosphorus hydrolase (OPH; EC 3.1.8.1), encoded by the identical opd genes from soil bacterium Pseudomonas diminuta MG and Flavobacterium sp

  • We proposed the activation of DFP hydrolysis by aminoalcohols and kinetically addressed this hypothesis

  • The results indicate that all the examined aminoalcohols activate the OPH-catalyzed hydrolysis of DFP

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Summary

Introduction

Organophosphorus hydrolase (OPH; EC 3.1.8.1), encoded by the identical opd genes from soil bacterium Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551, has been characterized as a typical phosphotriesterase (PTE) with a rather broad substrate specificity [1,2,3,4]. This enzyme, in the form of native or recombinant protein, has been documented to catalyze the hydrolysis of a wide variety of acetylcholinesterase (AchE) inhibitors, the chemical.

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