FIGURE 2. Concentration dependence of the observed rate on ETVTP concentration for WT HIV-1 RT.

A, single-turnover incorporation was measured by mixing a preincubated solution of RT (250 nm) and DNA/DNA primer/template (50 nm) with various concentrations of ETVTP (curves displayed represent 0.5 μm (○), 1 μm (□), 5 μm (◇), 20 μm (●) and 50 μm (△)) and MgCl₂ (10 mm) at 37 °C (all concentrations are final after mixing). B, K_d,ETVTP curve was generated from all ETVTP concentrations tested (500 nm to 50 μm) where the observed rate (k_obsd) was plotted against ETVTP concentration. The fit to the data gives an equilibrium binding constant (K_d,ETVTP) of 2.23 ± 0.67 μm and a maximum rate of incorporation (k_pol) of 0.107 ± 0.007 s⁻¹. C, pre-steady-state burst kinetics of incorporation of ETVMP into a DNA/DNA primer/template by WT RT were measured by mixing a preincubated solution of RT (100 nm) and primer/template (300 nm) with ETVTP (50 μm) and Mg²⁺ (10 mm) under rapid quench conditions (all concentrations are final after mixing). The reaction was quenched at indicated times and analyzed by 20% sequencing gel electrophoresis. The solid line represents a fit to a burst equation with an amplitude (A) equal to 79 ± 12 nm, an observed first-order rate constant for the burst phase (k_obsd) equal to 0.125 ± 0.031 s⁻¹, and an observed rate for the linear phase (k_ss) equal to 0.020 ± 0.006 s⁻¹.