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Chemical Research in Toxicology Sep 2019The single residue mutation of butyrylcholinesterase (BChE) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal...
The single residue mutation of butyrylcholinesterase (BChE) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.
Topics: Biocatalysis; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Echothiophate Iodide; Hydrolysis; Isoflurophate; Kinetics; Molecular Dynamics Simulation; Mutation; Paraoxon; Protein Binding; Protein Engineering; Thermodynamics
PubMed: 31411024
DOI: 10.1021/acs.chemrestox.9b00146 -
Molecules (Basel, Switzerland) Mar 2020Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol...
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 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 and 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 = 0.20 ± 0.03 mM and = 5.4 ± 1.6 min. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency ( = 2.6 ± 0.2 mM; = 53400 min). With a = (2.6 ± 1.6) × 10 Mmin, 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.
Topics: Biocatalysis; Butyrylcholinesterase; Caulobacteraceae; Echothiophate Iodide; Enzymes; Humans; Hydrolysis; Kinetics; Molecular Docking Simulation; Mutant Proteins; Organophosphorus Compounds; Phosphoric Triester Hydrolases; Spectrometry, Fluorescence; Sulfolobus
PubMed: 32192230
DOI: 10.3390/molecules25061371