Figure 6.

Kinetics of PP_iinhibition on dATP incorporation.A, fluorescence measurement during PP_i inhibition on dATP incorporation. T7 DNA polymerase E514Cou (500 nM), thioredoxin (10 μM), 27/45 DNA (600 nM), and PP_i (0–8 mM) were mixed with dATP (50 μM) and Mg²⁺ (12.5 mM) to start the reaction. Data at each concentration were fit to a double exponential function (black lines). B, rate versus PP_i concentration for inhibition of dATP incorporation. Rates are from data in (A). Data are shown for the fast phase (red, k_obs 1) and slow phase (green, k_obs 2), fit to a line and a hyperbola, respectively (black lines). The slope of the linear fit of the slow phase derived from the fitting is −0.028 ± 0.003 mM⁻¹ s⁻¹, which suggests weak competitive inhibition by PP_i for binding to the enzyme. The hyperbolic fit to the slow phase gives an apparent K_d,PPi: 775 ± 75 μM. Inset: Amplitude versus PP_i concentration for the slow phase, estimating an apparent K_d of 350 ± 80 μM. C, quench flow measurement of PP_i inhibition of dATP incorporation. T7 DNA polymerase E514Cou (120 nM), thioredoxin (2.4 μM), FAM-27/45 DNA (200 nM), BSA (0.1 mg/ml), and PP_i (0–12 mM) from one syringe were mixed with dATP (20 μM) and Mg²⁺ (12.5 mM) from the other syringe to start the reaction. Data at each PP_i concentration are shown fit to a burst equation (black lines). D, rate versus PP_i for inhibition on dATP incorporation. Rates are from data in (C). Data for the exponential phase of the burst at each concentration are shown fit to a hyperbola (black line) and gives an apparent K_i,PPi: 5.9 ± 3.1 mM.