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Steady-state kinetics of enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus as monitored by spectrofluorimetry

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dc.contributor.author Zueva I.V.
dc.contributor.author Lushchekina S.V.
dc.contributor.author Daudé D.
dc.contributor.author Chabrière E.
dc.contributor.author Masson P.
dc.date.accessioned 2021-02-25T20:56:15Z
dc.date.available 2021-02-25T20:56:15Z
dc.date.issued 2020
dc.identifier.uri https://dspace.kpfu.ru/xmlui/handle/net/162724
dc.description.abstract © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 ± 0.03 mM and kcat = 5.4 ± 1.6 min−1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53,400 min−1). With a kcat/Km = (2.6 ± 1.6) × 107 M−1min−1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P–S bonded OPs by thiol-free OP hydrolases.
dc.subject Calbiochem Probe IV
dc.subject Cholinesterase
dc.subject Echothiophate
dc.subject Organophosphate hydrolase
dc.subject Phosphotriesterase
dc.subject P–S bonded organophosphorus agents
dc.subject QM/MM
dc.title Steady-state kinetics of enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus as monitored by spectrofluorimetry
dc.type Article
dc.relation.ispartofseries-issue 6
dc.relation.ispartofseries-volume 25
dc.collection Публикации сотрудников КФУ
dc.source.id SCOPUS-2020-25-6-SID85082076285


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  • Публикации сотрудников КФУ Scopus [24551]
    Коллекция содержит публикации сотрудников Казанского федерального (до 2010 года Казанского государственного) университета, проиндексированные в БД Scopus, начиная с 1970г.

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