Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Figures & data

Scheme 1.  Paraoxon hydrolysis.

Figure 1.  Typical gel filtration of Co-PTE and Zn-PTE. The size of Zn- or Co-PTE in solution was determined by gel filtration with an initial enzyme concentration of 1 mg/mL and a volume sample of 200 μL. A gel filtration of Co-PTE is shown. The absorbance profiles of Co-PTE (bold dashed line) and molecular weight standards (thin line) were recorded at 280 nm. The molecular weight of β-lactoglobulin (14.4 kDa), ovalbumin (43 kDa) and bovine serum albumin (67 kDa), and the estimated value of Co-PTE (36 kDa) are indicated at the top of each corresponding peaks.

Figure 2.  X-Ray Fluorescence spectrum of recombinant Zn-PTE and Co-PTE. XRF spectrum for energy between 5 and 10 KeV of 0.1 mM Zn-PTE (a) and 0.1 mM Co-PTE (b).

Figure 3.  Stability of Zn-PTE and Co-PTE under assay conditions. Paraoxon hydrolysis of Zn-PTE and Co-PTE in the absence of additives, with 0.1% of β-lactoglobulin, with 50 μM ZnCl₂ and with both additives, 5 minutes after dilution of the stock enzyme.

Figure 4.  Michaelis-Menten Plot of paraoxon hydrolysis by Zn-PTE and Co-PTE. Initial reaction rate plotted against substrate concentration for paraoxon hydrolysis by PTE purified with Zn²⁺(○) and Co²⁺(□).

Table 1.  Paraoxon hydrolysis catalyzed by recombinant wild type PTE expressed in the presence of zinc and cobalt chloride and mutants expressed in the presence of cobalt chloride.

	kcat (s^−1)*	Km (mM)	kcat/Km (M^−1s^−1)	Kiapp (μM)	n	Partial factor (a)
Zn-PTE	1290±10	0.26±0.01	5×10^6	155±10	2	0.50±0.01
Co-PTE	4010±40	0.40±0.02	10×10^6	52±6	1	0.45±0.02
Co-His123Asn	1610±30	0.34±0.03	4.7×10^6	230±50	1	0.30±0.05
Co-His123Ala	390±5	0.39±0.03	1×10^6	300±100	1	0.47±0.07
Co-His123Ile	50±1	0.36±0.03	0.14×10^6	630±50	1	0

* Determined from V_max = k_cat[E_t], assuming that 100% of enzyme molecules are active; these values are therefore the minimal values of k_cat.

Figure 5.  Inhibition of paraoxon hydrolysis of Co-PTE and Zn-PTE by ZnCl₂. Reaction rate versus paraoxon concentration for Zn-PTE (a) and Co-PTE (b) in the absence (○) and presence of 0.1 mM (□), 0.25 mM (×) and 0.5 mM (+) ZnCl₂. Lineweaver-Burk plot of Zn-PTE (c) and Co-PTE (d) activities in the absence (○) and presence of 0.1 mM (□), 0.25 mM (×) and 0.5 mM (+) ZnCl₂. Dixon plots of Zn-PTE (e) and Co-PTE (f) with0.28 mM (○), 0.42 mM (□), 0.56 mM (×), 0.7mM (+), 3.5 mM (△) paraoxon. The best-fit curves or lines were determined using GOSA-fit and Equation (2), with n=2 for Zn-PTE and n=1 for Co-PTE.

Figure 6.  Inhibition of Co-PTE-catalyzed hydrolysis of an analogue of VX by ZnCl₂2. Hydrolysis of an analogue of VX by Co-PTE in the presence of increasing concentrations of ZnCl₂ up to 2 mM.

Figure 7.  Crystal structure of PTE. Cartoon view of the crystal structure of PTE-Cd/Cd (pdb code 1PSC). α-Helixes are in red and β-sheets in yellow. The allosteric ligand EBP is represented in ball and stick. Residues from the putative Zn binding site and the catalytic site are in stick.