Fig 1.

Peroxidase activity of recombinant TpxD using the thioredoxin system as a reductant. The reaction mixture contained 100 mM HEPES-NaOH (pH 7.0), 0.2 mM NADPH, 3.4 mM E. coli Trx, 0.36 mM E. coli TrxR, 5 μM purified TpxD, and 1 mM H₂O₂. Reactions were carried out with a total volume of 0.2 ml at 25°C and were started by the addition of H₂O₂ to the mixture. (A) Each curve is an absorbance time course at 340 nm, due to NADPH oxidation. In control experiments, TpxD (blue), H₂O₂ (green), thioredoxin reductase (pink), or thioredoxin (violet) was omitted from the reaction mixture. The data shown are from one representative experiment done in duplicates, but it was repeated 3 times, with similar results. (B) Michaelis-Menten curve for the determination of K_m, V_max, and K_cat/K_m values, with H₂O₂ concentrations plotted on the x axis and velocity plotted on the y axis.