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Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry

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 Brev...

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Autores principales: Zueva, Irina V., Lushchekina, Sofya V., Daudé, David, Chabrière, Eric, Masson, Patrick
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144395/
https://www.ncbi.nlm.nih.gov/pubmed/32192230
http://dx.doi.org/10.3390/molecules25061371
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author Zueva, Irina V.
Lushchekina, Sofya V.
Daudé, David
Chabrière, Eric
Masson, Patrick
author_facet Zueva, Irina V.
Lushchekina, Sofya V.
Daudé, David
Chabrière, Eric
Masson, Patrick
author_sort Zueva, Irina V.
collection PubMed
description 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 K(m) = 0.20 ± 0.03 mM and k(cat) = 5.4 ± 1.6 min(−1). The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (K(m) = 2.6 ± 0.2 mM; k(cat) = 53400 min(−1)). With a k(cat)/K(m) = (2.6 ± 1.6) × 10(7) M(−1)min(−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.
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spelling pubmed-71443952020-04-13 Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry Zueva, Irina V. Lushchekina, Sofya V. Daudé, David Chabrière, Eric Masson, Patrick Molecules Article 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 K(m) = 0.20 ± 0.03 mM and k(cat) = 5.4 ± 1.6 min(−1). The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (K(m) = 2.6 ± 0.2 mM; k(cat) = 53400 min(−1)). With a k(cat)/K(m) = (2.6 ± 1.6) × 10(7) M(−1)min(−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. MDPI 2020-03-17 /pmc/articles/PMC7144395/ /pubmed/32192230 http://dx.doi.org/10.3390/molecules25061371 Text en © 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/).
spellingShingle Article
Zueva, Irina V.
Lushchekina, Sofya V.
Daudé, David
Chabrière, Eric
Masson, Patrick
Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry
title Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry
title_full Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry
title_fullStr Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry
title_full_unstemmed Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry
title_short Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry
title_sort steady-state kinetics of enzyme-catalyzed hydrolysis of echothiophate, a p–s bonded organophosphorus as monitored by spectrofluorimetry
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144395/
https://www.ncbi.nlm.nih.gov/pubmed/32192230
http://dx.doi.org/10.3390/molecules25061371
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