FIGURE 7.
GTP concentration dependence of the kinetics of correct nucleotide incorporation. A, time courses of GTP incorporation opposite CMP in template at various GTP concentrations are shown. A mixture of NS5pol (0.6 μm) and P12/T26 (0.5 μm) in reaction buffer were incubated at 37 °C for 1 h. Aliquots of this mixture were rapidly mixed with GTP (12.5 (▿), 25 (▴), 50 (△)100 (■), 200 (□), 400 (●), and 800 (○) μm after mixing) to start nucleotide incorporation reactions. The concentration of NS5pol and P12/T26 after mixing is 0.3 and 0.25 μm, respectively. The reactions were quenched at various time intervals. Each time course was fit to a two-exponential equation (solid lines). B, data within 1-s reaction time from A are shown to demonstrate the fitting of the fast phase. C, shown is GTP concentration dependence of the incorporation rate for the fast phase. The rates of the fast phase were plotted against GTP concentration, and the data were fit to a hyperbola, yielding a Kd of 275 ± 52 μm and a kpol of 18 ± 1 s−1.