Figure 2.

Pre-steady-state pulse–chase kinetics of dTTP hydrolysis. 4A′ (3.3 μM hexamer) was mixed with [α-³²P]dTTP (100–600 μM) in a rapid quench-flow instrument at 18°C, and the reactions were chased with dTTP or EDTA (see Materials and Methods). (A) Representative time course of the pulse–chase kinetics at 100 μM dTTP is shown. (Inset) The entire pulse–chase time course to 60 s. The pulse–chase kinetics fit best to two exponentials (solid line) followed by a linear phase. The first phase amplitude is 1.09 ± 0.05 dTTP per hexamer, fast exponential rate constant is 10.0 ± 1.1 s⁻¹, second phase amplitude is 0.61 ± 0.07 dTTP per hexamer, second exponential rate constant is 0.23 ± 0.07 s⁻¹, and steady-state dTTPase turnover is 0.06 ± 0.002 s⁻¹. The dotted line shows the poor fit to one exponential followed by a linear equation. (B) The [dTTP] dependence of the first phase amplitude (▴) and rate constant [EDTA chase data (•) and dTTP chase data (♦)] are shown. The linear [dTTP] dependence of the fast exponential rate constant provided a k_on of 0.07 ± 0.01 mM⁻¹⋅s⁻¹ (slope) and a k_off of 0.4 ± 1.1 s⁻¹ (y intercept).