TABLE 2.
The thermodynamics of cofactors binding to wild-type and to its C298S mutant hAR (pH 6.5) as a function of temperature
ITC experiments conducted for the NADPH binding at 15 °C revealed weak binding, which is supported by the close to zero binding enthalpy, i.e. −38.9 to −4.00 (15–25) = 0, produced by extrapolating the binding enthalpy of −38.9 kJ/mol for NADPH at 25 °C down to 15 °C. The uncertainties are S.D. of the average value.
| Protein | Cofactor | Kb | −ΔbG | −ΔbH | TΔbS |
|---|---|---|---|---|---|
| m−1 | kJ mol−1 | kJ mol−1 | kJ mol−1 | ||
| At 15 °C | |||||
| Wild-type | NADP+ | 16.7 ± 2.1 × 106 | 39.8 ± 0.3 | 17.7 ± 1.1 | 22.1 ± 1.1 |
| C298S | NADP+ | 11.6 ± 1.1 × 106 | 34.0 ± 0.3 | 21.0 ± 2.9 | 13.0 ± 2.9 |
| At 25 °C | |||||
| Wild-type | NADP+ | 9.2 ± 2.1 × 106 | 39.7 ± 0.6 | 36.3 ± 1.4 | 3.4 ± 1.5 |
| C298S | NADP+ | 10.4 ± 1.2 × 106 | 40.1 ± 0.3 | 40.2 ± 0.7 | 0 |
| Wild-type | NADPH | 6.6 ± 1.6 × 106 | 38.9 ± 0.6 | 32.8 ± 3.2 | 6.1 ± 3.3 |
| C298S | NADPH | 3.0 ± 1.3 × 106 | 37.0 ± 1.1 | 36.7 ± 3.8 | 0 |
| At 35 °C | |||||
| Wild-type | NADP+ | 7.6 ± 1.0 × 106 | 40.6 ± 0.3 | 68.2 ± 5.7 | −27.6 ± 5.7 |
| C298S | NADP+ | 8.0 ± 0.7 × 106 | 40.7 ± 0.2 | 64.6 ± 2.5 | −23.9 ± 2.5 |
| Wild-type | NADPH | 7.8 ± 0.2 × 106 | 40.7 ± 0.1 | 51.2 ± 5.4 | −10.5 ± 5.4 |
| C298S | NADPH | 1.1 ± 0.3 × 106 | 35.7 ± 0.4 | 79.5 ± 9.8 | −43.8 ± 9.8 |