FIGURE 4.
Pre-steady state kinetics of correct single nucleotide incorporation of EFdA-TP and dATP by HIV-1 RT. Preincubated HIV-1 RT (50 nm) and Td31/Pd18 (A) or Td26/Pd18+5 (50 nm) (B) were mixed by a quench-flow instrument with various concentrations of EFdA-TP or dATP for reaction times varying between 5 ms and 2 s. The amount of product at different reaction times was fit to the burst equation (Equation 1) to obtain burst phase rates of nucleotide incorporation. Observed rate constants were then plotted using a hyperbolic equation (Equation 3) to estimate kpol and Kd(dNTP) (A and B). The sequences of the T/Ps are shown below the plots, and the calculated constants are shown in C. Error bars represent S.D. of at least two independent experiments.