Table 1.
Kinetic parameters of wildtype and variant Ltp2–ChsH2DUF35 with 17-HOPC-CoA as substrate
The Y294F/Y344F variant has no detectable activity. The Y294F and G82P had very-low activity that precludes the determination of Km and kcat values. At the respective concentrations of 25 and 23 μm 17-HOPC-CoA substrates, Y294F variant had a specific activity of (5.3 ± 2.3) × 10−4 μmol·min−1·mg−1, and G82P variant had a specific activity of (5.8 ± 0.59) × 10−4 μmol·min−1·mg−1. For comparison, the Vmax of the wildtype enzyme is 7.4 ± 0.21 μmol·min−1·mg−1. ND means not determined; NA means no detectable activity.
| Enzyme | Km | kcat | kcat/Km |
|---|---|---|---|
| μm | s−1 | m−1 s−1 | |
| Wildtype | 2.4 ± 0.30 | 7.1 ± 0.20 | (3.0 ± 0.38) × 106 |
| H296A | (7.6 ± 0.98) × 10−1 | (1.7 ± 0.041) × 10−1 | (2.2 ± 0.29) × 105 |
| H346A | (6.2 ± 1.4) × 10−1 | (1.6 ± 0.081) × 10−1 | (2.6 ± 0.60) × 105 |
| Y344F | 2.4 ± 0.34 | (1.7 ± 0.062) × 10−2 | (7.1 ± 1.0) × 103 |
| Y294F | ND | ND | ND |
| Y294F/Y344F | NA | NA | NA |
| G82P | ND | ND | ND |