Table 3.
Impart of individual mutations on kinetic properties of engineered kinase
| R4.V3-[85] |
R4.V3-[172] |
R4.V3-[179] |
R4.V3-[193] |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 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 | 0.13 ± 0.01 | 92 ± 14 | 1.4 | 13.9 ± 0.9 | 43 ± 9 | 326 | 0.22 ± 0.01 | 188 ± 29 | 1.2 | 0.08 ± 0.01 | 251 ± 36 | 0.3 |
| (−100) | (−34) | (−3438) | (−1.1) | (−22) | (−14) | (−58) | (−70) | (−4000) | (−161) | (−93) | (−16 000) | |
| dC | 0.13 ± 0.01 | 334 ± 65 | 0.39 | 11.7 ± 0.5 | 4 ± 1 | 2925 | 0.15 ± 0.01 | 35 ± 3 | 4.3 | 0.05 ± 0.01 | 105 ± 15 | 0.46 |
| (−90) | (−167) | (−15 000) | (1) | (−2) | (−2) | (−78) | (−18) | (−1360) | (−234) | (−53) | (−12 700) | |
| dA | <0.01 | >3000 | – | 12.4 ± 0.7 | 218 ± 36 | 57 | 0.25 ± 0.05 | 3000 ± 500 | 0.08 | <0.01 | >3000 | – |
| – | – | – | (−1.2) | (−2) | (−2.5) | (−63) | (−31) | (−2000) | – | – | – | |
| dG | <0.01 | >3000 | – | 4.3 ± 0.1 | 721 ± 22 | 6 | 0.06 ± 0.02 | 1500 ± 200 | 0.04 | 0.05 ± 0.01 | 1100 ± 243 | 0.05 |
| – | – | – | (−3) | (−1.6) | (−4.5) | (−200) | (−3) | (−700) | (−246) | (−2.5) | (−560) | |
| ddT | 1.36 ± 0.01 | 49 ± 3 | 28 | 0.49 ± 0.03 | 186 ± 28 | 2.6 | 0.48 ± 0.01 | 124 ± 15 | 3.9 | 0.84 ± 0.02 | 154 ± 16 | 5.5 |
| (+2.6) | (+2.3) | (+6) | (−1.1) | (−1.6) | (−1.8) | (−1.1) | (−1.1) | (−1.2) | (+1.7) | (−1.3) | (+1.2) | |
| fddT | 0.03 ± 0.002 | 81 ± 17 | 0.4 | 0.10 ± 0.01 | 14 ± 3 | 7.5 | 0.04 ± 0.01 | 14 ± 1 | 2.5 | 0.02 ± 0.01 | 10 ± 1 | 2.5 |
| (−6) | (+1.7) | (−3) | (−1.8) | (+10) | (+5.7) | (−4.5) | (+10) | (−2) | (−9) | (+14) | (−2) | |
Kinetic parameters of R4.V3 with natural 2′-deoxyribonucleosides (T, dC, dA, dG) and nucleoside analogs (ddT, fddT) after reverse engineering the four mutations. Numbers in parenthesis are fold change in catalytic efficiency for the particular substrate (kcat/KM [variant]/kcat/KM [DmdNK]).