Figure 4.

Kinetics of pyrophosphorolysis.A, hand quench pyrophosphorolysis with 3’-[α- ³²P]-labeled DNA. A solution of T7 DNA polymerase E514Cou (500 nM), thioredoxin (10 μM), BSA (0.1 mg/ml), 3’-[α-³²P]-28/45 DNA (100 nM), and PP_i (1–12 mM) was mixed with unlabeled dATP (20 μM) and Mg²⁺ (12.5 mM) to start the reaction. Data at each PP_i concentration are shown fit to a single exponential function (black lines). B, rate versus PP_i concentration for pyrophosphorolysis experiment. Rates are from the data in (A). Data are shown fit to a hyperbola (black line), estimating an apparent K_d,PPi: 5.6 ± 1.2 mM and k_PPi 0.20 ± 0.02 s⁻¹. C, stopped flow pyrophosphorolysis T7 DNA polymerase E514Cou (500 nM), thioredoxin (10 μM), 28/45 DNA (600 nM), and PP_i (1–8 mM) were mixed with Mg²⁺ (12.5 mM) to start the reaction. Data at each PP_i concentration were fit to a single exponential function (black lines). D, rate and amplitude versus PP_i concentration for stopped flow pyrophosphorolysis. Rates and amplitudes are from data in (C). Data are shown fit to a hyperbola (black line) estimating an apparent K_d,PPi: 950 ± 390 μM. Inset: Amplitude versus PP_i concentration. Shown fit to a hyperbola (black line), estimating an apparent K_d of 2.2 ± 0.3 mM. Increasing amplitude with increasing PP_i concentration is indicative of a "conformational selection" mechanism.