TABLE 1.
Parameters of wild-type and mutant GOOX
Steady state kinetic data are the means ± standard errors of at least three independent experiments. The values of the transient kinetic data were extracted from the hyperbolic fit to raw data of typically three to five independent measurements. In the case of kred, the standard deviation of the measurements at substrate saturating conditions is used as an error indicator. E°′ is the mid-point potential for the flavin cofactor determined at pH 7.0 and 25 °C shown as the means ± standard deviation of three independent experimental set-ups. Because of the unusual observations upon evaluation of the redox potentials for H70A, only an approximate value is presented.
| Wild type | H70A | C130A | |
|---|---|---|---|
| kcat (s−1) | 11.90 ± 0.2 | 0.22 ± 0.01 | 0.58 ± 0.025 |
| Km (mm) | 0.061 ± 0.005 | 0.22 ± 0.01 | 0.40 ± 0.02 |
| kcat/Km (s−1m m−1) | 194.3 ± 12.2 | 1.01 ± 0.07 | 1.50 ± 0.03 |
| Kd (mm) | 0.21 ± 0.03 | 0.32 ± 0.02 | 6.4 ± 0.6 |
| kred (s−1) | 170 ± 20 | 1.0 ± 0.1 | 23 ± 2 |
| E°′ (mV) | 126 ± 2 | ∼69 | 61 ± 1 |
| Tm (°C) | 56 | 48 | 52 |
| [Guanidine HCl]½ (m) | 3.2 | 1.9 | 2.4 |