Fig 1.
Molecular structures of DFP, GB, GD, diethyl fluorophosphate, and paraoxon (ethyl-paraoxon).
Fig 2.
OPH-catalyzed hydrolysis of DFP.
Fig 3.
Molecular structures of aminoalcohols, aminoalcohol analogs, amines and alcohols examined in this study.
Fig 4.
Kinetic analysis of OPH-catalyzed hydrolysis of DFP in the presence of 300 mM aminoalcohols.
The reaction was carried out in the presence of 100 μM ZnCl2, at pH 8.0 and 25°C. The initial enzyme concentration, [E], is 10 nM for every reaction. The initial velocity (V) (A) and the kcat (V/[E]) (B) are respectively plotted against DFP concentrations (0, 2, 4, 6, 8, 10, 20, 30, 40, and 50 mM). Solid line represents the best fit of the Michaelis-Menten equation using the Enzyme Kinetics Module from GraphPad Prism (version 5.0). Symbols for the buffers: control (in the absence of aminoalcohols), ○; monoethanolamine (MEA), □; diethanolamine (DEA), △; and triethanolamine (TEA), ▽. Data are expressed as the mean±SD of three independent experiments.
Fig 5.
Kinetic analysis of OPH-catalyzed hydrolysis of DFP with increasing triethanolamine (TEA) concentrations.
The reaction was carried out in the presence of 100 μM ZnCl2, at pH 8.0 and 25°C. The initial enzyme concentration, [E], is 10 nM for every reaction. The initial velocity (V) (A) and the kcat (V/[E]) (B) are respectively plotted against DFP concentrations (0, 2, 4, 6, 8, 10, 20, 30, 40, and 50 mM). Solid line represents the best fit of the Michaelis-Menten equation using the Enzyme Kinetics Module from GraphPad Prism (version 5.0). Symbols for TEA concentration (mM): 0, ○; 100, □; 200, △; 300, ▽; and 400 ◇. Data are expressed as the mean±SD of three independent experiments.
Table 1.
Kinetic parameters of OPH in the DFP hydrolysis in the presence of aminoalcohols.
Table 2.
Kinetic parameters of OPH in the DFP hydrolysis in the presence of 300 mM alcohols.
Table 3.
Kinetic parameters of OPH in the DFP hydrolysis in the presence of amines.
Table 4.
Changes in kinetic parameters of OPH towards DFP hydrolysis after adding a mixture of alcohol and amine.
Table 5.
Changes of kinetic parameters for OPH-catalyzed hydrolysis of DFP after adding analog molecules of MEA, DEA and TEA.
Table 6.
Kinetic parameters of OPH mutants in the DFP hydrolysis with or without triethanolamine (TEA).
Fig 6.
Kinetic analysis for EDTA inhibition of OPH activities towards DFP in the addition of 0 mM (A) and 300 mM (B) triethanolamine (TEA).
Except for the TEA and EDTA concentrations particularly mentioned, the reaction conditions and statistics analysis were the same as described in Fig 4.
Fig 7.
Kinetic analysis of fluoride inhibition of OPH-catalyzed DFP hydrolysis in the absence of triethanolamine.
The reaction was carried out in the presence of 100 μM ZnCl2, at pH 8.0 and 25°C. The initial enzyme concentration, [E], is 10 nM for every reaction. The initial velocity (V) (A) and the kcat (V/[E]) (B) at 0, 1, 2, and 3 mM NaF are respectively plotted against DFP concentrations (0, 2, 4, 6, 8, 10, 20, 30, 40, and 50 mM). Solid line represents the best fit of the Michaelis-Menten equation using the Enzyme Kinetics Module from GraphPad Prism (version 5.0). Symbols for NaF concentration (mM): 0, ○; 1, □; 2, △; and 3, ▽. Data are expressed as the mean±SD of three independent experiments.
Fig 8.
Kinetic analysis of fluoride inhibition of OPH-catalyzed DFP hydrolysis in the presence of triethanolamine.
The reaction was carried out in 0 mM (○), 100 mM (□), 200 mM (△), and 300 mM (▽) TEA-HCl buffer (pH 8.0 and 25°C) containing 100 μM ZnCl2 and 3 mM NaF. The initial enzyme concentration, [E], is 10 nM for every reaction. The initial velocity (V) (A) and the kcat (V/[E]) (B) are respectively plotted against DFP concentrations (0, 2, 4, 6, 8, 10, 20, 30, 40, and 50 mM). Solid line represents the best fit of the Michaelis-Menten equation using the Enzyme Kinetics Module from GraphPad Prism (version 5.0). Data are expressed as the mean±SD of three independent experiments.
Table 7.
Kinetic parameters of wild-type OPH in the DFP hydrolysis in fluoride inhibition with or without triethanolamine.
Fig 9.
Analysis of inhibition kinetic constant (Ki) for fluoride (NaF) in OPH-catalyzed hydrolysis of DFP.
The inhibition analysis of fluoride was performed at the increasing triethanolamine (TEA) concentrations: 0 mM (A), 100 mM (B), 200 mM (C) and 300 mM (D). The TEA-HCl buffer (pH 8.0) at 25°C contained 100 μM ZnCl2, the initial enzyme of 10 nM and 0–3 mM NaF. At each TEA concentration, the 1/V was plotted against 1/[DFP] under 0 mM (●), 1 mM (□), 2 mM (■), and 3 mM (○) NaF conditions. Inset shows the replot of the apparent Km obtained from the double-reciprocal plot versus inhibitor (NaF) concentrations. Each point in the plot is the average of triplicate determination with the experimental error less than 10%.
Fig 10.
Effect of triethanolamine on inhibition of OPH-catalyzed hydrolysis of DFP by fluoride (NaF) given at a later reaction stage.
The velocity (V) for OPH-catalyzed hydrolysis of DFP under 0 (○) and 300 mM (□) TEA condition was determined once per minute with adding of 3 mM NaF (inhibitor) at the end of the first 5 minutes, i.e. at the beginning of 6th minute in total reaction time (10 minutes). The DFP concentration used was 20 mM, and the other reaction conditions and statistics analysis were the same as described in Fig 4. The black arrows (up and down) both indicated the time point to add NaF with final concentration (3 mM), and the dotted vertical line in the diagram indicated the first time-point to make record after NaF adding.
Fig 11.
Effect of adding aminoalcohole at a later stage (6–10 minutes) of OPH-catalyzed hydrolysis of DFP.
The velocity (V) for OPH-catalyzed hydrolysis of DFP was determined once per minute with adding 0 (○), 100 (□), 200 (△), and 300 mM (▽) TEA at the end of the first 5 minutes, i.e. at the beginning of 6th minute in total 10 minutes reaction time. The DFP concentration used was 20 mM, and the other reaction conditions and statistics analysis were the same as described in Fig 4. The black arrow (down) indicated the time point to add TEA with defined final concentrations, and the dotted vertical line in the diagram indicated the first time-point to make record after TEA adding.