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 muta...
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MDPI AG
2020-03-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/6/1371 |
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doaj-1877f7b4dc9d4778b4070156ccf7206c2020-11-25T02:10:43ZengMDPI AGMolecules1420-30492020-03-01256137110.3390/molecules25061371molecules25061371Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by SpectrofluorimetryIrina V. Zueva0Sofya V. Lushchekina1David Daudé2Eric Chabrière3Patrick Masson4Arbuzov Institute of Organic and Physical Chemistry, Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”, Arbuzov str. 8, 420088 Kazan, RussiaEmanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin str 4, 119334 Moscow, RussiaGene&GreenTK, HU Méditerranée Infection, Jean Moulin Blvd 19–21, 13005 Marseille, FranceAix-Marseille University, IRD, APHM, MEPHI, IHU-Méditerranée Infection, 15005 Marseille, FranceKazan Federal University, Neuropharmacology Laboratory, Kremlevskaya str 18, 480002 Kazan, RussiaEnzyme-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 <i>Brevundimonas diminuta</i> 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 <i>Brevundimonas diminuta</i> and <i>Sulfolobus solfataricus</i> 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 <i>K<sub>m</sub></i> = 0.20 ± 0.03 mM and <i>k<sub>cat</sub></i> = 5.4 ± 1.6 min<sup>−1</sup>. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (<i>K<sub>m</sub></i> = 2.6 ± 0.2 mM; <i>k<sub>cat</sub></i> = 53400 min<sup>−1</sup>). With a <i>k<sub>cat</sub>/K<sub>m</sub></i> = (2.6 ± 1.6) × 10<sup>7</sup> M<sup>−1</sup>min<sup>−1</sup>, 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.https://www.mdpi.com/1420-3049/25/6/1371p–s bonded organophosphorus agentsechothiophatecalbiochem probe ivorganophosphate hydrolasephosphotriesterasecholinesteraseqm/mm |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Irina V. Zueva Sofya V. Lushchekina David Daudé Eric Chabrière Patrick Masson |
| spellingShingle |
Irina V. Zueva Sofya V. Lushchekina David Daudé Eric Chabrière Patrick Masson Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry Molecules p–s bonded organophosphorus agents echothiophate calbiochem probe iv organophosphate hydrolase phosphotriesterase cholinesterase qm/mm |
| author_facet |
Irina V. Zueva Sofya V. Lushchekina David Daudé Eric Chabrière Patrick Masson |
| author_sort |
Irina V. Zueva |
| title |
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_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_sort |
steady-state kinetics of enzyme-catalyzed hydrolysis of echothiophate, a p–s bonded organophosphorus as monitored by spectrofluorimetry |
| publisher |
MDPI AG |
| series |
Molecules |
| issn |
1420-3049 |
| publishDate |
2020-03-01 |
| 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 <i>Brevundimonas diminuta</i> 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 <i>Brevundimonas diminuta</i> and <i>Sulfolobus solfataricus</i> 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 <i>K<sub>m</sub></i> = 0.20 ± 0.03 mM and <i>k<sub>cat</sub></i> = 5.4 ± 1.6 min<sup>−1</sup>. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (<i>K<sub>m</sub></i> = 2.6 ± 0.2 mM; <i>k<sub>cat</sub></i> = 53400 min<sup>−1</sup>). With a <i>k<sub>cat</sub>/K<sub>m</sub></i> = (2.6 ± 1.6) × 10<sup>7</sup> M<sup>−1</sup>min<sup>−1</sup>, 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. |
| topic |
p–s bonded organophosphorus agents echothiophate calbiochem probe iv organophosphate hydrolase phosphotriesterase cholinesterase qm/mm |
| url |
https://www.mdpi.com/1420-3049/25/6/1371 |
| work_keys_str_mv |
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