Table 2.
Kinetic data for purified XPF–ERCC1 mutants.
| Protein | Mutant | Mutation rationale | Vmax (fmol min−1) | Km (nM) | kcat (s−1) | kcat/Km (nM−1 min−1) |
|---|---|---|---|---|---|---|
| XPF | WT | 104.7 ± 2.8 | 12.1 ± 1.4 | 20.9 ± 0.66 | 1.73 | |
| XPF | L230R | FA | 65 ± 2.1 | 8.1 ± 1.1 | 13.0 ± 3.3 | 1.60 |
| XPF | L236R | FA/CS | 91.2 ± 2.5 | 10.8 ± 1.2 | 18.2 ± 0.5 | 1.69 |
| XPF | E239K | FA | 88.9 ± 4.7 | 12.4 ± 2.1 | 16.9 ± 0.7 | 1.36 |
| XPF | S786F | ICLR deficient | 80.9 ± 3.8 | 32.5 ± 1.5 | 7.9 ± 0.8 | 0.24 |
| XPF | 323–326 Δ | ICLR deficient | 120.2 ± 1.4 | 20.5 ± 0.8 | 24.2 ± 0.3 | 1.18 |
| XPF | S312A | Autoinhibition | 126.1 ± 28.2 | 5.1 | 13.5 ± 1.8 | 2.65 |
| XPF | W274A, H275A | Autoinhibition | 193.4 ± 8.6 | 15.2 ± 2.4 | 38.7 ± 1.3 | 2.55 |
| XPF | R112A | DNA binding | 24.5 ± 1.1 | 4.32 ± 0.63 | 5.6 ± 0.2 | 1.30 |
| XPF | 829–833 Δ | Autoinhibition linker disruption | 20.2 ± 0.5 | 1.9 ± 1.2 | 3.8 ± 0.2 | 2.00 |
| XPF | R589W | XP | 8.22 ± 2.6 | 25.9 ± 6.1 | 1.4 ± 0.9 | 0.05 |
| ERCC1 | L253A | Autoinhibition | 33.1 ± 2.7 | 28.7 ± 22.3 | 3.4 ± 0.3 | 0.12 |
Each kinetic value was obtained from 3 technical replicates (n = 3) ± standard deviation (SD).