Table 1 |.
Apparent kinetics parameters of Bs GDH variants
| Enzyme | Cofactor | Km (mM) | kcat (s−1) | kcat/Km (mM−1s−1) |
|---|---|---|---|---|
| Bs GDH WT (wild type) | NAD+ | (3.0 ± 0.1) × 10−2 | 5.5 ± 1.1 | (1.8 ± 0.3) × 102 |
| NADP+ | (1.5 ± 0.1) × 10−2 | 4.3 ± 0.1 | (2.8 ± 0.1) × 102 | |
| NMN+ | NDa | NDa | (4.7 ± 0.5) × 10−4 | |
| Bs GDH Triple (I195R-A93K-Y39Q) | NAD+ | 3.7 ± 0.9 | (4.1 ± 0.3) × 10−1 | (1.1 ± 0.2) × 10−1 |
| NADP+ | (6.1 ± 1.5) × 10−1 | 4.4 ± 0.1 | 7.5 ± 1.8 | |
| NMN+ | 6.4 ± 0.8 | 3.1 ± 0.1 | (5.1 ± 0.6) × 10−1 | |
| Bs GDH Ortho (I195R-A93K-Y39Q-S17E) | NAD+ | 6.5 ± 1.3 | (2.5 ± 0.2) × 10−2 | (3.8 ± 0.5) × 10−3 |
| NADP+ | 2.0 ± 0.1 | (2.2 ± 1.3) × 10−2 | (1.1 ± 0.8) × 10−2 | |
| NMN+ | 5.9 ± 1.0 | 1.2 ± 0.1 | (2.1 ± 0.2) × 10−1 |
Reactions were performed in 35 mM Tris-HCl buffer (pH 8.0) at 25 °C using a constant glucose concentration of 140 mM. Detailed rate equations can be found in the Methods. Errors shown are the standard deviation of three independent measurements. ND, not determined.
a
Enzyme could not be saturated with the cofactor concentrations tested.