Table 2.
Kinetic constants (Km, μM; kcat, s−1; and kcat/Km, s−1 mM−1) of wild-type LiPH8 and its single and multiple variants oxidizing VA, DMP and ABTS (means and 95% confidence limits).
| Wild-type LiPH8LiPH8 | Mutated variants |
|||||
|---|---|---|---|---|---|---|
| D165N | E168Q | E250Q | D264N | |||
| VA | Km | 73.0 ± 3.9 | 80.9 ± 4.1 | 348.0 ± 37.0 | 221.0 ± 22.0 | 90.1 ± 5.5 |
| kcat | 20.9 ± 0.2 | 12.5 ± 0.2 | 17.1 ± 0.7 | 5.2 ± 0.1 | 20.0 ± 0.3 | |
| kcat/Km | 282.0 ± 15.0 | 155.0 ± 8.0 | 49.2 ± 5.6 | 23.5 ± 2.4 | 222.0 ± 14.0 | |
| DMP | Km | 38.1 ± 5.8 | 42.7 ± 2.4 | 50.2 ± 3.9 | 39.3 ± 3.2 | 33.4 ± 1.0 |
| kcat | 9.9 ± 0.4 | 5.3 ± 0.1 | 7.0 ± 0.2 | 1.5 ± 0.0 | 10.1 ± 0.1 | |
| kcat/Km | 260.0 ± 41.0 | 214.0 ± 7.0 | 139.0 ± 12.0 | 38.2 ± 3.1 | 302.0 ± 10.0 | |
| ABTS | Km | 26.7 ± 1.2 | 30.3 ± 0.6 | 26.4 ± 1.8 | 37.2 ± 4.1 | 14.5 ± 0.7 |
| kcat | 19.9 ± 0.3 | 15.7 ± 0.1 | 16.2 ± 0.3 | 9.2 ± 0.3 | 22.5 ± 0.4 | |
| kcat/Km | 745.0 ± 35.0 | 520.0 ± 3.0 | 614.0 ± 43.0 | 247.0 ± 28.0 | 1550.0 ± 80.0 | |
| Mutated variants |
||||||
|---|---|---|---|---|---|---|
| F267I | D165N/E168Q | E168Q/E250Q | D264N/F267I | D165N/E168Q/D264N/F267I | ||
| VA | Km | 233.0 ± 20.0 | 368.0 ± 34.0 | 2160.0 ± 470.0 | 273.0 ± 24.0 | 2550.0 ± 628.0 |
| kcat | 15.7 ± 0.3 | 11.6 ± 0.3 | 7.6 ± 0.6 | 13.5 ± 0.3 | 20.7 ± 1.8 | |
| kcat/Km | 67.3 ± 6.0 | 31.5 ± 3.0 | 3.5 ± 0.8 | 49.4 ± 4.4 | 8.1 ± 2.1 | |
| DMP | Km | 64.2 ± 10.3 | 78.3 ± 10.5 | 75.4 ± 11.4 | 78.3 ± 6.8 | 81.8 ± 12.3 |
| kcat | 7.3 ± 0.4 | 9.6 ± 0.4 | 5.3 ± 0.3 | 9.2 ± 0.5 | 10.9 ± 0.5 | |
| kcat/Km | 114.0 ± 19.0 | 123.0 ± 17.0 | 70.3 ± 11.3 | 245.0 ± 22.0 | 133.0 ± 21.0 | |
| ABTS | Km | 28.8 ± 1.2 | 38.3 ± 3.4 | 5.4 ± 0.5 | 22.6 ± 1.9 | 5.9 ± 0.8 |
| kcat | 19.4 ± 0.2 | 26.2 ± 0.7 | 13.2 ± 0.3 | 29.3 ± 0.7 | 15.2 ± 0.8 | |
| kcat/Km | 674.0 ± 29.0 | 684.0 ± 63.0 | 2460.0 ± 220.0 | 1290.0 ± 120.0 | 2610.0 ± 390.0 | |