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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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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. |
format | Online Article Text |
id | pubmed-7144395 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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