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Role of Some Metal Ions on Steady–state Kinetics of Engineered Wild–type and Manganese (II) Binding Site Mutants of Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3)

Usenobong F. Ufot, Aniefiok E. Ite, Idorenyin H. Usoh, Monday I. Akpanabiatu

American Journal of Medical and Biological Research. 2016, 4(3), 42-52 doi:10.12691/ajmbr-4-3-2
  • Figure 1A. Ribbon Model showing the overall fold of engineered MnP3 from Phlebia radiata. α-Helices and heme are shown in red, β-strands in white, distal and proximal calcium in green, manganese in pink. Image generated using Web Lab Viewer (Raswin) and the heme, Mn, and Ca obtained from crystal structure of versatile peroxidase from Pleurotuseryngiideposited in the protein Data Bank, entry code 3FJWA
  • Figure 1B. A model of manganese (II) binding site, with its associated amino acids ligands, of engineered P. radiata MnP3. The Mn (II) (pink) at the edge of the heme propionate is surrounded by ligands, Glu40, Glu44 and Asp186.Image generated using WebLab Viewer (Accelrys) and the heme, Mn, and ligands obtained from crystal structure of versatile peroxidase from Pleurotuseryngii deposited in the protein Data Bank, entry code 3FJWA
  • Figure 2. Steady-state Kinetic Parameters for (A) Mn (II) and (B) ABTS Oxidation by Wild-type and Mutant MnP3 Enzymes from Phlebia radiata.Each data point represents the mean of three independent determinations with standard errors indicated. Assays were carried out as described in materials and methods. For assays with Mn (II), a fixed concentration of 0.1 mM H2O2 was used and the data fitted to the Michaelis equation using Sigma Plot. For ABTS studies, a fixed concentration of 0.01 µM was used for all assays involving the wild-type enzyme while that of the mutant variants range from 0.1 – 6.5 mM. The kinetic parameters extracted from the plots are presented in Table 1
  • Figure 3. The Dependence of Turnover Number on (A) Mn (II) and (B) ABTS Concentration in the Presence of Monovalent and Divalent Ions. () MnSO4 only, () 0.8 mM NaSO4, () 0.8 mM ZnSO4, () 0.2 mM CuSO4, () 0.8 mM CoSO4. Reaction mixtures contained 100 mM Na tartrate buffer, pH 5.0 at 25°C and fixed H2O2 concentration (0.1 mM). Varying concentrations of MnSO4 and ABTS were used for figures A and B respectively as stated in the material and methods section. Apparent kinetic parameters were estimated from the best fit to the Michaelis-Menten equation
  • Figure 4. Effect of metal ion addition on Mn (II) oxidation of rPr-MnP3 mutant enzymes (E40H, E44H and E40H/E44H. () 0.8 mM ZnSO4, () 0.2 mM CuSO4, () 0.8 mM CoSO4 () 0.8 mM NaSO4. The activity assay performed in 100 mM Na tartrate, pH 5.0 at 25°C using varying concentrations MnSO4 and fixed concentration 0.1 mM H2O2
  • Figure 5. Plots showing effect of metal ion effectors Mn (II) oxidation by rPr-MnP3 mutant enzyme, D186H. () 0.8 mM ZnSO4, () 0.2 mM CuSO4, and () 0.8 mM CoSO4, and () 0.8 mM NaSO4 (as control) Activity assays performed in 100 mM Na tartrate, pH 5.0 at 25°C using varying concentrations MnSO4 and fixed H2O2 concentration (0.1mM)