TABLE 1.
Pre-steady-state kinetic constants for the incorporation of dTTP and AZTTP into heteropolymeric template/primer by wild-type and mutant RTs
The template/primer 31T/21P was used as the substrate. Data shown are the mean values ± S.D. Each of the assays was performed independently at least three times. Inter-assay variability was below 20%. All determinations were carried out under single turnover conditions.
| RT | Nucleotide | kpol | Kd | kpol/Kd | Selectivitya |
|---|---|---|---|---|---|
| s−1 | μm | μm−1s−1 | |||
| WT (NL4-3) | dTTPb | 17.4 ± 2.0 | 7.1 ± 2.5 | 2.47 ± 0.93 | 0.47 ± 0.20 |
| AZTTP | 22.1 ± 1.6 | 18.9 ± 3.6 | 1.17 ± 0.24 | ||
| Δ67/T69G/K70R | dTTP | 22.4 ± 1.4 | 13.2 ± 2.7 | 1.70 ± 0.36 | 1.15 ± 0.32 (2.4) |
| AZTTP | 8.6 ± 0.3 | 4.4 ± 0.8 | 1.95 ± 0.36 | ||
| Δ67/T69G/K70R/K219E | dTTP | 19.9 ± 0.8 | 5.5 ± 1.1 | 3.62 ± 0.74 | 1.00 ± 0.31 (2.1) |
| AZTTP | 9.4 ± 0.4 | 2.6 ± 0.6 | 3.61 ± 0.80 | ||
| Δ69 | dTTPb | 12.3 ± 1.0 | 14.8 ± 3.0 | 0.83 ± 0.18 | 0.55 ± 0.17 (1.2) |
| AZTTP | 14.0 ± 1.1 | 30.6 ± 6.2 | 0.46 ± 0.10 | ||
| Δ69/K219E | dTTP | 12.4 ± 1.3 | 14.8 ± 5.1 | 0.83 ± 0.29 | 0.28 ± 0.13 (0.6) |
| AZTTP | 9.8 ± 1.0 | 42.4 ± 11.9 | 0.23 ± 0.07 | ||
| S68G/Δ69/K70G | dTTP | 11.1 ± 0.5 | 10.3 ± 1.4 | 1.08 ± 0.15 | 0.42 ± 0.11 (0.9) |
| AZTTP | 16.4 ± 1.6 | 35.4 ± 7.3 | 0.46 ± 0.10 | ||
| S68G/Δ69/K70G/K219E | dTTP | 10.8 ± 0.9 | 11.0 ± 3.2 | 0.97 ± 0.30 | 0.30 ± 0.14 (0.6) |
| AZTTP | 9.7 ± 1.1 | 32.8 ± 11.0 | 0.29 ± 0.10 |
a The selectivity is the ratio of [kpol/Kd (AZTTP)]/[kpol/Kd (dTTP)]. Resistance (shown between parentheses) was calculated as the selectivity value obtained with each mutant enzyme divided by the selectivity obtained with the WT RT.
b Data were taken from Kisic et al. (24).