Table 1.
Kinetic and thermodynamic parameters of nucleotide interactions with WT and mutant SecA.
| Parameter | Ligand | Method | Temp. | WT BsSecA | BsSecA E208Q | WT EcSecA | EcSecA E210Q |
|---|---|---|---|---|---|---|---|
| n | Mg-ADP | ITC | 25 °C | 0.89 | 0.92 | 0.93 | -- |
| ΔH° (kcal mol−1) | Mg-ADP | ITC | 25 °C | −13.00 ± 0.07 | −14.0 ± 0.1 | −20.0 ± 0.1 | -- |
| ΔS° (cal mol−1 K−1)a | Mg-ADP | ITC | 25 °C | −13.0 ± 0.3 | −24.0 ± 0.3 | −34.0 ± 0.4 | -- |
| ΔG° (kcal mol−1) | Mg-ADP | ITC | 25 °C | −9.20 ± 0.04 | −6.90 ± 0.02 | −9.80 ± 0.04 | -- |
| Kd (nM) | Mg-ADP | ITC | 25 °C | 180 ± 10 | 7700 ± 300 | 62 ± 4 | -- |
| kcat (min−1)b | Mg-ATP | M.G.c | 25 °C | 7.2 ± 0.6 | 0.24 ± 0.18d | 0.480 ± 0.024 | 0.120 ± 0.006d |
| Kd (nM) | Mg-MANT-ADP | Anisotropy | 25 °C | 160 ± 40e | 3500 ± 140 | 12 ± 3 | 120 ± 11 |
| Kd (nM) | Mg-MANT-ATP | Anisotropy | 25 °C | 190 ± 50f | 3800 ± 120 | 22 ± 7f | 120 ± 9 |
| Kd (nM) | Mg-MANT-ADP | Anisotropy | 5°C | 250 ± 30 | 460 ± 10 | 17 ± 7 | 16 ± 9 |
| koff (min−1) | Mg-MANT-ATP | Anisotropy | 5°C | 0.120 ± 0.003f | 32 ± 3 | 0.0400 ± 0.0004f | 5.6 ± 0.6 |
| koff (min−1) | Mg-MANT-ADP | Anisotropy | 25 °C | 1.1 ± 0.1 | -- | 0.070 ± 0.003 | -- |
| koff (min−1) | Mg-MANT-ATP | Anisotropy | 25 °C | 0.80 ± 0.03f | -- | 0.0500 ± 0.0007f | -- |
| kon (M−1 min−1) | Mg-MANT-ADP | Anisotropy | 25 °C | 11.0 ± 0.9 × 106 | -- | 18.0 ± 0.9×106 | 120.0 ± 0.9×106 |
| kon (M−1 min−1) | Mg-MANT-ATP | Anisotropy | 25 °C | 5.0 ± 0.6 × 106 | -- | 6.0 ± 0.6×106 | 40.0 ± 0.4 × 106 |
| Kd = koff/kon (nM) | Mg-MANT-ADP | Anisotropy | 25 °C | 100 ± 20 | -- | 4.0 ± 0.4 | -- |
| Kd = koff/kon (nM) | Mg-MANT-ATP | Anisotropy | 25 °C | 150 ± 20f | -- | 8 ± 1f | -- |
| Tm (°C) | none | DSC | N/A | 40.0 ± 0.1, 49.0 ± 0.1 | 27.0 ± 0.1, 41.0 ± 0.0 | 41.00 ± 0.03, 44.00 ± 0.05 | 39.0 ± 0.1, 44.0 ± 0.1 |
| Tm (°C) | Mg-ADP | DSC | N/A | 47.0 ± 0.4, 51.0 ± 0.4 | 32.00 ± 0.03, 41.00 ± 0.03 | 47.00 ± 0.03 | 41.0 ± 0.1, 44.0 ± 0.1 |
Calculated by the ORIGIN ITC module from ΔG° = ΔH° − TΔS°, with ΔG° being calculated from the optimized value of Kd from curve-fitting the ITC data.
Previous research has shown that kcat is equal to koff for Mg-ADP release 13, meaning that Mg-ADP release is rate limiting in the basal ATP hydrolysis cycle of SecA. The kcat values reported here for Mg-ADP do not match the koff values reported below for Mg-MANT-ADP because the fluorescently derivatized nucleotide binds to SecA tighter and is released more slowly than Mg-ADP itself.
Measured via Malachite Green phosphate-release assay calibrated with inorganic phosphate.
This level of ATP turnover may reflect the activity of contaminating enzymes. See text.
This value was obtained using 900 nM [Mg-MANT-ADP]. WT BsSecA showed evidence of a modest reduction in Kd value in experiments conducted at substantially lower nucleotide and therefore protein concentrations, suggesting that dissolution of the BsSecA dimer may increase nucleotide-binding affinity slightly.
Mg-MANT-ATP affinities and off-rates for the wild-type enzymes reflect the release rates of Mg-MANT-ADP.