Table 3.
Binding parameters for the interaction of WT and mutated CaFADSs with RF, FMN, FAD and ATP, as determined by ITC (n = 3; means ± SD).
| K d (µM) | ||||||
|---|---|---|---|---|---|---|
| FADS:RF | FADS:FMN | FADS:FAD | FADS:ATP | FADS:ADP:FMN | ||
| 10 mM MgCl2 | FMNAT site | RFK site | ||||
| WT a | 24.1 (2) ± 3.6 | 7.8 (1) ± 0.9 | 0.74 (1) ± 0.10 | 30.2 (2) ± 4.5 | 0.04 ± 5 10−3 | 0.90 ± 0.10 |
| K202A | 31.4 (1) ± 4.7 | 12.2 (≪1) ± 1.0 | 6.4 (≪1) ± 1.0 | 64.5 (1) ± 16.9 | 0.01 ± 1.5 10−3 | 1.4 ± 0.3 |
| E203A | 10.3 (1) ± 2.3 | 7.2 (1) ± 1.6 | 61.8 (1) ± 15.2c | 43.4 (2) ± 9.3 | 0.94 ± 0.20 | 9.12 ± 1.4c |
| F206A | n.d.b | 6.8 (1) ± 3.8 | 1.9 (≪1) ± 0.1 | 15.3 (2) ± 2.4 | 1.2 ± 0.7c | 9.71 ± 2.7c |
| F206K | 7.6 (2) ± 2.5 | 3.1 (≪1) ± 0.5 | 11.5 (1) ± 1.7 | 29.1 (2) ± 8.0 | 0.10 ± 0.03 | 0.39 ± 0.06 |
| F206W | 10.3 (1) ± 4.0 | 3.6 (≪1) ± 1.1 | 0.68 (≪1) ± 0.30 | 26.5 (2) ± 11.0 | 0.05 ± 0.01 | 0.85 ± 0.30 |
| D298A | 5.3 (2) ± 1.1 | 18.8 (1) ± 2.9c | 3.0 (≪1) ± 0.3 | 32.9 (2) ± 2.7 | 1.2 ± 0.2c | 12.09 ± 2.1c |
| D298E | 17.9 (2) ± 8.5 | 19.1 (1) ± 1.8c | 3.7 (≪1) ± 0.2 | 46.2 (2) ± 14.5 | 1.4 ± 0.3c | 3.4 ± 0.6 |
| V300A | 22.6 (≪1) ± 1.7 | 2.5 (≪1) ± 0.5 | 2.2 (≪1) ± 1.0 | 31.6 (2) ± 9.1 | 0.10 ± 0.005 | 3.1 ± 0.5 |
| V300K | n.d.b | n.d.b | 4.0 (1) ± 0.5 | 15.3 (2) ± 2.4 | 0.22 ± 0.03 | 2.2 ± 0.4 |
| E301A | 58.0 (2) ± 8.4 | 5.9 (1) ± 0.7 | 2.4 (1) ± 0.9 | 60.7 (2) ± 9.5 | 0.35 ± 0.07 | 4.7 ± 0.6 |
| E301K | 142 (2) ± 42c | 13.6 (1) ± 1.2 | 2.9 (1) ± 0.4 | 60.2 (2) ± 10.1 | 0.19 ± 0.04 | 13.2 ± 1.9c |
| L304A | 1.2 (1) ± 0.4 | 18.3 (1) ± 0.8c | 28.2 (1) ± 3.3c | 17.6 (1) ± 1.0 | 0.51 ± 0.20 | 1.1 ± 0.3 |
| L304K | 40.6 (2) ± 7.7 | 37.1 (1) ± 3.0c | 26.2 (1) ± 1.0c | 39.6 (2) ± 2.5 | 1.7 ± 0.1c | 2.2 ± 0.5 |
The stoichiometry for the interaction is shown in brackets. For those showing a stoichiometry of N = 2, data correspond to an average K d (K d,av) of two independent binding sites. Data obtained at 25 °C in 20 mM PIPES pH 7.0, 10 mM MgCl2.
bDespite these variants exhibiting different spectra upon mixing with the ligand, no heat exchange was detected in the corresponding ITC titration, indicating a very low binding enthalpy.
cValues showing statistically significant differences from the WT, as determined by the one-way ANOVA test (P < 0.002; n = 3, confidence interval 95%).