Skip to main content
. 2020 Oct 5;295(50):17265–17280. doi: 10.1074/jbc.RA120.015557

Figure 9.

Figure 9.

dATP (correct nucleotide) burst kinetics at 4 °C for WT and E514Cou T7 DNA polymerase by rapid quench. A, pre-steady-state burst kinetics for WT T7 DNA polymerase. WT T7 DNA polymerase (60 nm), thioredoxin (1.2 µm), BSA (0.1 mg/ml), and FAM-27/45-18T DNA (125 nm) were mixed with Mg2+ (12.5 mm) and varying concentrations of dATP (5–110 µm) in a KinTek RQF-3 quench-flow instrument, and the reaction was quenched with EDTA from a quench syringe. Data for each dATP concentration are shown in different colors. The solid lines through the data points are the best fit obtained by fitting the data globally in KinTek Explorer using the model in Scheme 2. The best-fit values derived from the fitting are kpol: 181 ± 25 s−1 and Kd,app: 28 ± 8 µm. B, pre-steady-state burst kinetics for T7 DNA polymerase E514Cou. T7 DNA polymerase E514Cou (75 nm), thioredoxin (1.5 µm), BSA (0.1 mg/ml), and FAM-27/45-18T DNA (150 nm) were mixed with Mg2+ (12.5 mm) and varying concentrations of dATP (2.5–110 µm) in the quench-flow to start the reaction and then with EDTA to stop the reaction at various times. Data for each dATP concentration are shown in different colors. The solid lines are the best global fit of the data using KinTek Explorer with the model in Scheme 2 to derive the parameters kpol: 202 ± 14 s−1 and Kd,app: 15 ± 3 µm.