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

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 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.

1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM/MM Studies

“…Kinetic and molecular modeling studies showed that Probe IV does not interfere with activity measurements so that ATC-based assays using DTNB and Probe IV are correlated. The experimental conditions of assay in a Peltier thermostated spectrofluorimeter F-7100 (Hitachi Ltd., Tokyo, Japan) were previously decribed [21,22]. Molecular docking was performed using as targets several structures of hAChE co-crystallized with different non-covalent inhibitors (PDB ID 4EY4-4EY8 [15]) and one covalently bound to an organophosphorus adduct (human AChE phosphonylated by sarin, PDB ID 5FPQ, [23]).…”

“…Thus, after 20 min inhibition, the system was diluted 10-and 1000-fold to drop TFK concentration, and the enzyme activity was monitored as a function of time. Because after dilution, the enzyme activity became very low for accurate monitoring of activity vs. time, instead of the classical Ellman assay, the activity was monitored using a new method with the fluorescent thiol probe (Calbiochem Probe IV) instead of DTNB [21,22]. Results showed that dilution of TFK speeded up the deacylation process (blue and green curves in Figure 6, showing faster recovery of enzyme activity).…”

Enzymatic Decontamination of G-Type, V-Type and Novichok Nerve Agents

“…GG1 and GG2 were engineered from Brevundimonas diminuta PTE (P0A434) [ 44 ] and were produced as previously described [ 45 ]. In short, pET22b-GG1 and pET22b-GG2 were transformed in E. coli BL21(DE 3 )-pGro7/GroEL (TaKaRa) chaperone expressing strain.…”

“…This study aims to demonstrate (i) that enzyme-based formulations can be used to decontaminate a broad spectrum of nerve agents including the poorly studied Novichok agents and (ii) that such formulations can be used to degrade high concentrations of neurotoxic chemicals used in solution or spread on surfaces. To this end, we evaluate the potential of two engineered PTEs, namely GG1 and GG2, developed by the company Gene&GreenTK [ 44 , 45 ]. We first demonstrate the outstanding efficacy of the solution to degrade a wide spectrum of OPs including four insecticides and six OPNA surrogates, rapidly and completely.…”

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions

“…Thus, most researchers concentrate their efforts on genetic modification of the microenvironment of His and other amino acid residues. However, this artificial progress is now rather modest [ 78 ], and natural evolving is much more creative [ 79 , 80 ] ( Figure 17 ).…”