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

Irina Zueva (1) , Sofya Lushchekina (2) , David Daudé (3, 4) , Eric Chabriere (5, 3) , Patrick Masson (6)

1 Kazan Scientific Centre of the Russian Academy of Sciences
2 Emanuel Institute of Biochemical Physics
3 IHU Marseille - Institut Hospitalier Universitaire Méditerranée Infection
4 Gene&GreenTK
5 MEPHI - Microbes évolution phylogénie et infections
6 KFU - Kazan Federal University

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 = 53400 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.

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

Domaines: Bactériologie; Cardiologie et système cardiovasculaire; Maladies émergentes; Maladies infectieuses; Parasitologie; Virologie

Irina Zueva, Sofya Lushchekina, David Daudé, Eric Chabriere, Patrick Masson. Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry. Molecules, 2020, 25 (6), pp.1371. ⟨10.3390/molecules25061371⟩. ⟨hal-02517839⟩