Table 1.
Michaelis-Menten Kinetic Parameters of Mutant CKB Enzymes.1
| CKB Mutant | kcat (s−1) | Km (mM) | kcat/Km (M−1s−1) | % WT kcat/Km (M−1s−1) |
|---|---|---|---|---|
| WT | 15.5 ± 0.7 | 2.05 ± 0.4 | 7550± 1560 | 100 |
| C74S† | 15.4 ± 1.2 | 1.23 ± 0.5 | 12500±5040 | 166 |
| R96P† | 12.1 ± 1.1 | 9.11 ± 2.5 | 1340 ±381 | 17.6 |
| S128R† | 12.0 ± 0.8 | 4.16 ± 1.0 | 2890 ± 696 | 38.3 |
| R132H† | N/A | N/A | 159 ± 12 | 2.1 |
| R172G | 14.2 ± 0.5 | 1.71 ± 0.3 | 8350± 1460 | 111 |
| R172P† | 17.2 ± 1.0 | 1.77 ± 0.5 | 9760 ± 2670 | 129 |
| R236Q† | 13.7 ± 1.5 | 14.7 ± 4.2 | 927±281 | 12.3 |
| D268H | 17.3 ± 0.9 | 1.13 ± 0.3 | 15300 ±4080 | 203 |
| C283S† | 13.1 ± 1.3 | 17.2 ± 4.2 | 761 ±201 | 10.1 |
| R292Q† | 13.0 ± 1.6 | 14.6 ± 4.6 | 887±301 | 11.8 |
| H296P | 14.7 ± 0.9 | 7.04 ± 1.4 | 2090 ± 424 | 27.7 |
| H296R† | 17.8 ± 2.9 | 30.8 ± 9.9 | 579 ± 208 | 7.67 |
| P300L | 14.0 ± 0.7 | 1.66 ± 0.4 | 8460 ±1880 | 112 |
| T327R | 15.9 ± 0.8 | 1.69 ± 0.4 | 9460 ± 2300 | 125 |
| I361T | 17.4 ± 0.7 | 2.02 ± 0.4 | 8580±1600 | 114 |
| E368G | 17.6 ± 1.0 | 1.96 ± 0.5 | 8990 ± 2340 | 119 |
1
Mutant CKB assays were performed with n = 3, while WT CKB was performed n = 8, and data were globally fit to the Michaelis-Menten equation. Fitted kinetic parameters are shown with standard error. The data obtained from R132H better fit a linear regression, thus kcat and Km values could not be determined and are reported as N/A (not applicable).
†:
mutants that displayed reduced formation of TFV-DP in the in vitro activity assay