Table 2.
Kinetic properties of wild-type and engineered kinases
|
DmdNK |
R4.V1 (T85M, S123L, A147V, E172V, D189E, H193Y, N210D, S223G) |
R4.V3 (T85M, E172V, Y179, H193Y) |
R4.V9 (T85M, S123L, N130D, E172V, H193Y, L2031) |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| kcat/KM | kcat/KM | kcat/KM | kcat/KM | |||||||||
| kcat (s−1) | KM (μM) | (103 × s−1 M−1) | kcat (s−1) | KM (μM) | (103 × s−1 M−1) | kcat (s−1) | KM (μM) | (103 × s−1 M−1) | kcat (s−1) | KM (μM) | (103 × s−1 M−1) | |
| T | 12.9 ± 0.9 | 2.7 ± 0.5 | 4813 | 0.09 ± 0.01 | 286 ± 27 | 0.3 | 0.03 ± 0.003 | 319 ± 88 | 0.1 | 0.08 ± 0.01 | 111 ± 14 | 0.7 |
| (−143) | (−106) | (−16 000) | (−430) | (−118) | (−48 000) | (−160) | (−41) | (−6875) | ||||
| dC | 11.7 ± 0.7 | 2.0 ± 0.4 | 5850 | 0.09 ± 0.01 | 156 ± 7 | 0.6 | 0.03 ± 0.002 | 95 ± 19 | 0.36 | 0.11 ± 0.01 | 46 ± 13 | 2.3 |
| (−130) | (−78) | (−9750) | (−390) | (−48) | (−16 250) | (−106) | (−23) | (−2543) | ||||
| dA | 15.8 ± 0.4 | 98 ± 3 | 161 | 0.08 ± 0.01 | 1560 ± 346 | 0.05 | <0.01 | >3000 | – | 0.16 ± 0.03 | 1734 ± 619 | 0.09 |
| (−197) | (−16) | (−3220) | – | – | – | (−99) | (−18) | (−1789) | ||||
| dG | 12.3 ± 0.4 | 450 ± 54 | 28 | 0.04 ± 0.01 | 1180 ± 300 | 0.03 | <0.04 | >3000 | – | 0.19 ± 0.01 | 1545 ± 154 | 0.12 |
| (−307) | (−2.6) | (−933) | – | – | – | (−65) | (−3.4) | (−233) | ||||
| ddT | 0.53 ± 0.03 | 115 ± 22 | 4.6 | 0.27 ± 0.02 | 102 ± 33 | 2.6 | 0.49 ± 0.01 | 167 ± 9 | 3 | 0.24 ± 0.01 | 101 ± 21 | 2.4 |
| (−2) | (+1.1) | (−1.8) | (−1.1) | (−1.5) | (−1.5) | (−2.2) | (+1.1) | (−1.9) | ||||
| fddT | 0.18 ± 0.01 | 139 ± 24 | 1.3 | 0.020 ± 0.001 | 25 ± 3 | 0.8 | 0.021 ± 0.001 | 10 ± 1 | 1.9 | 0.017 ± 0.001 | 13 ± 2 | 1.3 |
| (−9) | (+5.6) | (−1.6) | (−9) | (+14) | (+1.5) | (−11) | (+11) | (1) | ||||
Kinetic parameters for natural 2′-deoxyribonucleosides (T, dC, dA, dG) and nucleoside analogs (ddT, fddT) of wild-type enzyme (DmdNK) and selected candidates from the directed evolution experiments after four rounds. Numbers in parentheses are fold change in catalytic efficiency for the particular substrate (kcat/KM [variant]/kcat/KM [DmdNK]).