FIGURE 2.
A, pH dependence of kcat for FdsABG. The reactions were performed at 30 °C in a buffer containing 75 mm of malate, K2HPO4, Tris, and glycine, brought to pH 5–10. The fit to the data (solid line) yielded two pKa values of 5.6 and 9.3, respectively. B, Lineweaver-Burk plots for the reaction of FdsABG with formate at the following [NAD+]: 1.62 mm (closed circles), 0.54 mm (open circles), 0.18 mm (closed triangles), and 0.06 mm (open triangles). C, Lineweaver-Burk plots for the reaction of FdsABG with NAD+ at the following [formate]: 3.24 mm (closed circles), 1.08 mm (open circles), 0.36 mm (closed triangles), and 0.12 mm (open triangles). D, secondary plots of 1/y intercept taken from B versus 1/[NAD+]. E, secondary plots of 1/y intercept taken from C. versus 1/[formate]. The reactions in B and C were performed in 75 mm K-PO4, pH 7.7, at 30 °C. Linear regression analysis of plots D and E yielded a KmNAD+ = 130 μm and Kmformate = 310 μm, respectively, and an average kcat of 201 s−1.