Fig. 2.

Effect of mutations in the P-loop-like sequence on nucleotide and PPi binding. Fluorescence intensity titration of the loop Trp is shown upon dUTPase binding to (A), dUPNPP (solid symbols) and dUDP (open symbols) (hDUT^R/K data shown in the inset separately for clarity) or (B), upon dUMP saturated dUTPase binding to PP_i. Signal intensities are normalized to the nucleotide-free state. Smooth lines through the data are hyperbolic fits yielding K_d values listed in Table 1. WT (squares), hDUT^R/K (triangles), and hDUT^ST/AA (left-pointing triangles) were used at 4 μM concentration. Error bars indicate SD for n = 3. Further evidences on nucleotide binding properties of the mutants are shown in the SI Results and Discussion.