Table 2.
The kinetic properties at 70°C of wild-type enzyme and mutant 13F2
| Substrate/cofactor | Wild-type AdhA | 13F2 | ||||
|---|---|---|---|---|---|---|
| K m (mM) | V max (U mg−1) | k cat/K m (s−1 mM−1) | K m (mM) | V max (U mg−1) | k cat/Km (s−1 mM−1) | |
| 2,5-hexanedione | 56.4 ± 0.6 | 1.9 ± 0.1 | 0.01 | 79.0 ± 1.7 | 2.9 ± 0.4 | 0.02 |
| Pyruvaldehyde | 1.2 ± 0.03 | 10.4 ± 0.2 | 3.8 | 4.2 ± 0.1 | 52.7 ± 0.5 | 5.4 |
| (2S,5S)-hexanediol | 5.4 ± 0.3 | 12.8 ± 0.4 | 1.0 | 327.6 ± 9.6 | 28.1 ± 1.1 | 0.04 |
| 2-pentanol | 30.3 ± 0.4 | 13.1 ± 0.8 | 0.1 | 51.1 ± 0.9 | 24.7 ± 2.1 | 0.2 |
| NADPHa | 0.4 ± 0.02 | 10.3 ± 0.07 | – | 0.1 ± 0.01 | 48.3 ± 0.9 | – |
| NADP+ | 0.2 ± 0.03 | 12.8 ± 0.2 | – | 0.7 ± 0.08 | 37.3 ± 1.0 | – |
All measurements were performed in duplicate
aThe kinetic parameters for NADPH and NADP+ were determined using, respectively, pyruvaldehyde and 2-pentanol as substrate