Table 1.
Specific Activities and Apparent Kinetic Constants of 6PGDHs for NMN+ and NADP+a
| NADP+ | NMN+ | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| enzyme | mutations | sp. act. (U/mg) | kcat (S−1) | KM (mM) | kcat/KM (mM−1 s−1) | sp. act. (U/mg) | kcat (S−1) | KM (mM) | kcat/KM (mM−1 s−1) |
| wild-type | - | 18.0 ± 0.8 | 15.9 ± 0.2 | 0.0012 ± 0.0001 | 13394.5 | 0.60 ± 0.01 | 1.3 ± 0.1 | 30.6 ± 1.7 | 0.042 |
| Mut 1–1 | R33I/T34I | 1.2 ± 0.2 | 14.8 ± 0.7 | 11.5 ± 1.1 | 1.3 | 0.68 ± 0.01 | 1.7 ± 0.1 | 37.9 ± 3.6 | 0.046 |
| Mut 2–1 | A11G/R33I/T34I | 10.2 ± 0.7 | 28.3 ± 0.6 | 2.1 ± 0.1 | 13.2 | 4.66 ± 0.02 | 10.2 ± 0.2 | 20.7 ± 0.7 | 0.49 |
| Mut 3–1 | A11G/R33I/T34I/D82L/T83L/Q86L | 16.4 ± 0.1 | 21.1 ± 0.3 | 0.39 ± 0.02 | 53.7 | 9.19 ± 0.09 | 19.3 ± 0.4 | 27.5 ±1.2 | 0.70 |
| Mut 4–1 | A11G/K27R/R33I/T34I/D82L/T83L/Q86L/I120F/D294V/Y383C/N387S/A447V | 20.0 ± 0.3 | 21.9 ± 0.2 | 0.22 ± 0.01 | 101.0 | 12.31 ± 0.14 | 18.9 ± 0.4 | 15.1 ± 0.6 | 1.25 |
| Mut 5–1 | A11G/K27R/R33I/T34I/F60Y/D82L/T83L/Q86L/K118N/I120F/D294V/F326S/Y383C/N387S/A447V | 20.6 ± 0.4 | 23.6 ± 0.3 | 0.21 ± 0.01 | 113.0 | 16.40 ± 0.40 | 25.8 ± 0.8 | 16.0 ± 1.1 | 1.62 |
| Mut 6–1 | A11G/K27R/R33I/T34I/F60Y//D82L/T83L/Q86L/K118N/I120F/D2S1E/D294V/F326S/F329Y/Y383C/N387S/V390G/A447V | 27.1 ± 0.8 | 28.9 ± 0.3 | 0.19 ± 0.01 | 148.6 | 17.70 ± 0.35 | 27.4 ± 0.5 | 13.5 ± 0.5 | 2.04 |
a
New introduced mutations relative to their parent enzyme are highlighted in bold; each value represents the average ± standard deviation of triplicate independent measurements