TABLE 2.
Kinetic constantsa describing the hydrolysis of the four canonical NTPs by the NS3 helicase derived from genotypes 1a, 1b, and 2a
| NTP and kinetic constants | Hel-1a | Hel-1b | Hel-2a |
|---|---|---|---|
| ATP | |||
| Stimulated kcat (s−1) | 54 ± 4 | 49 ± 5 | 58 ± 4 |
| Stimulated Km (μM) | 440 ± 90 | 230 ± 100 | 370 ± 90 |
| Basal kcat (s−1) | 1.4 ± 0.1 | 0.5 ± 0.1 | 1.8 ± 0.2 |
| Basal Km (μM) | 23 ± 9 | 5 ± 2 | 12 ± 4 |
| PolyU KNA (mM) | 1.0 ± 0.2 | 1.2 ± 0.5 | 0.77 ± 0.1 |
| GTP | |||
| Stimulated kcat (s−1) | 33 ± 2 | 41 ± 11 | 32 ± 2 |
| Stimulated Km (μM) | 1.4 ± 0.6 | 0.76 ± 0.1 | 0.98 ± 0.1 |
| Basal kcat (s−1) | 2.5 ± 0.5 | 1.3 ± 0.1 | 2.0 ± 0.6 |
| Basal Km (μM) | 45 ± 6 | 33 ± 10 | 45 ± 10 |
| Poly(U) KNA (mM) | 0.49 ± 0.2 | 1.1 ± 0.4 | 0.21 ± 0.05 |
| CTP | |||
| Stimulated kcat (s−1) | 29 ± 5 | 29 ± 4 | 36 ± 2 |
| Stimulated Km (μM) | 0.26 ± 0.1 | 0.16 ± 0.05 | 0.31 ± 0.06 |
| Basal kcat (s−1) | 1.4 ± 0.01 | 1.3 ± 0.5 | 1.6 ± 0.1 |
| Basal Km (μM) | 19 ± 5 | 5 ± 2 | 11 ± 2 |
| Poly(U) KNA (mM) | 0.45 ± 0.1 | 0.84 ± 0.03 | 0.35 ± 0.06 |
| UTP | |||
| Stimulated kcat (s−1) | 31 ± 3 | 35 ± 3 | 38 ± 3 |
| Stimulated Km (μM) | 0.36 ± 0.1 | 0.23 ± 0.04 | 0.49 ± 0.2 |
| Basal kcat (s−1) | 2.0 ± 0.2 | 1.2 ± 0.03 | 1.8 ± 0.4 |
| Basal Km (μM) | 28 ± 5 | 8 ± 4 | 17 ± 3 |
| Poly(U) KNA (mM) | 0.36 ± 0.05 | 0.70 ± 0.1 | 0.24 ± 0.07 |
a
Kinetic constants were determined for NS3 helicase isolated from genotype 1a (Hel-1a), genotype 1b (Hel-1b), and genotype 2a (Hel-2a). Each constant was determined from three separate titrations done at different enzyme concentrations. Errors represent the variation seen between separate experiments.