Table 2.
Apparent kinetic parameters for MtACK wild type and variants.
| Enzyme | NTP | Km (mM) | kcat (s−1) | kcat/Km (s−1·mM−1) | Km Acetate a (mM) |
|---|---|---|---|---|---|
| WT | ATP | 4.0 ± 0.3 | 715 ± 10 | 180 ± 9 | 14.3 ± 0.5 |
| - | CTP | 3.2 ± 0.4 | 460 ± 28 | 148 ± 13 | - |
| - | GTP | 7.2 ± 0.9 | 571 ± 22 | 80 ± 7 | - |
| - | TTP | 2.7 ± 0.1 | 540 ± 9 | 202 ± 2 | - |
| - | UTP | 2.7 ± 0.3 | 415 ± 19 | 152 ± 7 | - |
| - | ITP | 4.7 ± 0.2 | 742 ± 10 | 158 ± 5 | - |
| Gly331Ala | ATP | 10.2 ± 0.4 | 88 ± 1.8 | 8.9 ± 0.18 | 601 ± 8 |
| - | CTP | 15.7 ± 1.6 | 17 ± 0.8 | 1.1 ± 0.07 | - |
| - | GTP | 5.3 ± 0.2 | 67 ± 1.0 | 12.6 ± 0.40 | - |
| - | TTP | 11.2 ± 0.5 | 20 ± 0.5 | 1.8 ± 0.05 | - |
| - | UTP | * | 43 ± 8.1 | ND | - |
| - | ITP | 10.7 ± 0.2 | 84 ± 0.4 | 7.8 ± 0.17 | - |
| Gly331Gln | ATP | 18.4 ± 0.6 | 16 ± 0.2 | 0.9 ± 0.02 | ** |
| - | CTP | 11.1 ± 0.7 | 2.1 ± 0.05 | 0.2 ± 0.01 | - |
| - | GTP | 7.4 ± 0.4 | 8.6 ± 0.15 | 1.2 ± 0.04 | - |
| - | TTP | 12.1 ± 1.0 | 2.8 ± 0.10 | 0.2 ± 0.01 | - |
| - | UTP | 14.2 ± 0.5 | 2.8 ± 0.03 | 0.2 ± 0.01 | - |
| - | ITP | 10.1 ± 0.3 | 11 ± 0.1 | 1.0 ± 0.02 | - |
| Asn211Ala | ATP | 5.9 ± 0.5 | 374 ± 13 | 63.0 ± 5.7 | 165 ± 20 |
| Asn211Ser | ATP | 6.8 ± 0.6 | 13 ± 0.8 | 1.9 ± 0.1 | 140 ± 5 |
| Asn211Thr | ATP | 7.1 ± 0.3 | 32 ± 0.7 | 4.5 ± 0.2 | 470 ± 40 |
| Gly239Ala | ATP | 1.7 ± 0.1 | 77 ± 2.1 | 45.7 ± 2.6 | 113 ± 13.4 |
| Gly239Ser | ATP | 1.8 ± 0.1 | 6.8 ± 0.05 | 3.9 ± 0.19 | 94 ± 3.0 |
Notes: a Kinetic parameters for NTPs were determined at saturating acetate concentration when possible. The maximum acetate concentration used was 1 M. * Enzyme was not saturable for UTP. Kinetic parameters were determined with 25 mM UTP. ** Enzyme was not saturable for acetate.