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

Arbuzov Institute of Organic and Physical Chemistry, Federal Research Center “Kazan Scientific Center of the Russian Academy of Sciences”, Arbuzov str. 8, 420088 Kazan, Russia

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin str 4, 119334 Moscow, Russia

Gene&GreenTK, HU Méditerranée Infection, Jean Moulin Blvd 19–21, 13005 Marseille, France

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Aix-Marseille University, IRD, APHM, MEPHI, IHU-Méditerranée Infection, 15005 Marseille, France

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Kazan Federal University, Neuropharmacology Laboratory, Kremlevskaya str 18, 480002 Kazan, Russia

Author to whom correspondence should be addressed.

Molecules 2020, 25(6), 1371; https://doi.org/10.3390/molecules25061371

Received: 22 February 2020 / Revised: 14 March 2020 / Accepted: 16 March 2020 / Published: 17 March 2020

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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Abstract

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) × 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. View Full-Text

Keywords: P–S bonded organophosphorus agents; echothiophate; Calbiochem Probe IV; organophosphate hydrolase; phosphotriesterase; cholinesterase; QM/MM

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Zueva, I.V.; Lushchekina, S.V.; Daudé, D.; Chabrière, E.; Masson, P. Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry. Molecules 2020, 25, 1371. https://doi.org/10.3390/molecules25061371

AMA Style

Zueva IV, Lushchekina SV, Daudé D, Chabrière E, Masson P. Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry. Molecules. 2020; 25(6):1371. https://doi.org/10.3390/molecules25061371

Chicago/Turabian Style

Zueva, Irina V., Sofya V. Lushchekina, David Daudé, Eric Chabrière, and Patrick Masson. 2020. "Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry" Molecules 25, no. 6: 1371. https://doi.org/10.3390/molecules25061371

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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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