Figure 3.

Kinetics of dATP incorporation.A, quench flow pre-steady-state burst experiment. T7 DNA polymerase E514Cou (120 nM), BSA (0.1 mg/ml), thioredoxin (2.4 μM), and FAM-27/45 DNA (200 nM) were mixed with dATP (2.5–110 μM) and Mg²⁺ (12.5 mM) to start the reaction. Data at various dATP concentrations are shown as different colors, fit to a burst equation (black line) at each concentration. B, rate versus dATP concentration. Rates are from the fit of the exponential phase in (A) and are shown fit to a hyperbola (black line), estimating the maximum rate, k_pol = 210 ± 15 s⁻¹ and K_d,app =15.5 ± 2 μM. C, stopped flow dATP incorporation. T7 DNA polymerase E514Cou (750 nM), thioredoxin (15 μM), and DNA 27/45 (1 μM) were mixed with dATP (5–100 μM) and Mg²⁺ (12.5 mM) to start the reaction. Data at each dATP concentration were fit to a double exponential function (black lines). D, rate versus dATP concentration for stopped flow fluorescence. Rates are from the data in (C). The fast phase data (red points, k_obs 1) were fit to a hyperbola to estimate the maximum rate of the conformational change (k₂), 7800 ± 3400 s⁻¹. The slow phase data (green points, k_obs 2) were fit to a line, setting a lower limit on the maximum rate of the isomerization following chemistry (k₄) of 126 ± 4 s⁻¹.