Table 2.
Steady-state kinetic parameters for the FMNAT activity (FMN → FAD) of the different CaFADS variants (n = 3; means ± SE).
| k cat (min−1) | K m FMN (µM) | K m ATP (µM) | k cat/K m FMN (min−1 µM−1) | k cat/K m ATP (min−1 µM−1) | |
|---|---|---|---|---|---|
| WT | 5.5 ± 0.5 | 10.1 ± 1.0 | 22.4 ± 2.0 | 0.54 ± 0.07 | 0.25 ± 0.03 |
| K202A | 2.3 ± 0.2a | 2.9 ± 0.5a | 12.1 ± 2.9 | 0.80 ± 0.10 | 0.19 ± 0.05 |
| E203A | 3.2 ± 0.2a | 0.70 ± 0.10a | 10.8 ± 2.6 | 4.5 ± 0.7a | 0.30 ± 0.07 |
| F206A | 4.2 ± 0.2a | 2.9 ± 0.7a | 38.0 ± 7.2 | 1.45 ± 0.4 | 0.11 ± 0.02 |
| F206K | 6.2 ± 0.3 | 5.4 ± 0.6a | 38.8 ± 3.3 | 1.15 ± 0.1 | 0.16 ± 0.02 |
| F206W | 4.6 ± 0.2 | 1.7 ± 0.5a | 25.2 ± 6.1 | 3.3 ± 1 | 0.22 ± 0.05 |
| D298A | 6.1 ± 0.4a | 1.2 ± 0.2a | 20.7 ± 6.4 | 5.1 ± 0.9a | 0.30 ± 0.09 |
| D298E | 3.3 ± 0.3 | 0.95 ± 0.30a | 10.4 ± 3.3 | 3.47 ± 1.1 | 0.31 ± 0.10 |
| V300A | 4.9 ± 0.2 | 1.4 ± 0.2a | 46.2 ± 7.6a | 3.5 ± 0.5 | 0.11 ± 0.02 |
| V300K | 4.6 ± 0.3 | 8.3 ± 1.7 | 9.5 ± 1.4 | 0.55 ± 0.10 | 0.48 ± 0.08 |
| E301A | 6.9 ± 0.2 | 0.42 ± 0.02a | 19.7 ± 3.6 | 16.4 ± 0.9a | 0.35 ± 0.06 |
| E301K | 6.3 ± 0.5 | 0.85 ± 0.20a | 15.3 ± 3.6 | 7.4 ± 1.8a | 0.41 ± 0.10 |
| L304A | 2.5 ± 0.2a | 2.8 ± 0.3a | 34.7 ± 9.4 | 0.91 ± 0.10 | 0.07 ± 0.02 |
| L304K | 5.2 ± 0.5 | 15.2 ± 3.1a | 11.5 ± 1.8 | 0.34 ± 0.08 | 0.45 ± 0.08 |
Data obtained at 25 °C in 20 mM PIPES pH 7.0, 10 mM MgCl2.
aValues showing statistically significant differences from the WT, as determined by the one-way ANOVA test (P < 0.002; n = 3, confidence interval 95%).