FIG. 2.

Pre-steady-state kinetics of d4TMP incorporation by HIV-1 RT. (A) The incorporation of d4TMP (200 μM d4TTP) into the 22/45-mer DNA duplex (300 nM) by HIV-1 RT (86 nM) was determined at 37°C in buffer containing 10 mM MgCl₂. The time course for d4TMP incorporation by HIV-1 RT was fit to a burst equation as described previously (34), and the curve is drawn for an amplitude (A) equal to 86 ± 13 nM, k_obsd equal to 1.6 ± 0.4 s⁻¹, and a steady-state rate (r) equal to 14 ± 5 nM s⁻¹. k_ss (which is equal to r/A) was calculated to be 0.16 ± 0.05 s⁻¹. (B) The incorporation of d4TMP (200 μM d4TTP) into the 22/45-mer DNA/RNA heteroduplex (270 nM) by HIV-1 RT (89 nM) was determined at 37°C in buffer containing 10 mM MgCl₂. The time course for d4TMP incorporation by HIV-1 RT was fit to a burst equation, and the curve is drawn for an amplitude (A) equal to 89 ± 2 nM, k_obsd equal to 17 ± 2 s⁻¹, and a steady-state rate (r) equal to 9.5 ± 2.8 nM s⁻¹. k_ss (which is equal to r/A) was calculated to be 0.11 ± 0.03 s⁻¹.