Table 1.
Summary of pre-steady-state kinetic data of dNTP and 3′-NLa
| Nucleotide | kpol (s−1) | Kd,app (μM) | kpol/Kd,app | kpol/Kd,app (3′-AL/dATP) |
|---|---|---|---|---|
| WT dNTP | ||||
| dATP | 43.9 ± 8.5 | 1.3 ± 0.4 | 36.4 ± 9.1 | |
| dCTP | 15.4 ± 2.7 | 5.2 ± 0.7 | 2.9 ± 0.2 | |
| dTTP | 19.8 ± 1.4 | 4.4 ± 1.3 | 4.7 ± 1.2 | |
| dGTP | 50.9 ± 8.7 | 2.7 ± 0.6 | 20 ± 7.5 | |
| 3′-NL | ||||
| 3′-AL | 2.8 ± 0.6 | 49 ± 5.6 | 0.06 ± 0.02 | 0.00165 |
| 3′-CL | 2.6 ± 0.2 | 57.4 ± 17.3 | 0.05 ± 0.01 | |
| 3′-TL | 2.5 ± 1 | 258.6 ± 8.3 | 0.01 ± 0.004 | |
| 3′-GL | 6.4 ± 2.2 | 175.3 ± 31.4 | 0.04 ± 0.01 | |
| Y409A | ||||
| dATP | 1.6 ± 0.4 | 1 ± 0.4 | 1.6 ± 0.4 | |
| 3′-AL | 0.05 ± 0.01 | 61 ± 7 | 0.001 ± 0.0001 | 0.00062 |
| D542E | ||||
| dATP | 2.7 ± 0.1 | 30.4 ± 3.2 | 0.1 ± 0.01 | |
| 3′-AL | 0.03 ± 0.003 | 118.1 ± 19 | 0.0003 ± 0.00002 | 0.003 |
aThe experiment was performed by adding 1 μM 9°N-I to the incorporation reaction in the presence of 2 mM MgSO4 (see the Methods section). The ± values stand for standard deviation based on three fitted lines for kpol and Kd,app from three distinct aliquots of nucleotide samples (n = 3)