Characterization of enzymatic activity of Tk-PTP.

<p>Phosphatase activity assays were carried out at 20°C and 60°C as described in the Materials and Methods section. (A–B) Enzymatic reactions were carried out using 100 μM DiFMUP for 1 h with 10 nM purified recombinant Tk-PTP proteins in the 100 μL reaction buffer at pH 5.0 (<i>A</i>) or at the indicated pH levels (<i>B</i>). Tk-PTP, but not Tk-PTP(C93S), has dephosphorylating activity (<i>A</i>), which is optimum at pH 4.5–5.0 (<i>B</i>). (C) Kinetic parameters of 11 types of Tk-PTP proteins are listed. Enzymatic reactions were carried out with purified recombinant Tk-PTP proteins in 100 μL reaction buffer (pH 5.0). Production of DiFMU was detected by measuring fluorescence at 2 min intervals for 10 min, with DiFMUP concentrations of 50, 100, 250, 500, 1000, and 1500 μM. Initial velocity data at each substrate concentration were obtained by detecting the release of DiFMU between 2 and 10 min after the start of reaction, which was calculated from the slope of the each progress curve. Using these data, Michaelis-Menten curves shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197635#pone.0197635.s006" target="_blank">S6 Fig</a> were obtained using the program OriginPro 8.0, by fitting the initial velocities against each DiFMUP concentrations to the hill equation with hill coefficient of 1. Subsequently, Lineweaver-Burk plots shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197635#pone.0197635.s007" target="_blank">S7 Fig</a> were interpreted for the determination of V_max, <i>k</i>_cat and <i>K</i>_M values.