Table 3.
Rescue of hmt-tRNAPhe G34A aminoacylation efficiency with PheRS variants
| Hmt-PheRS | Hmt-tRNAPhe WT |
Hmt-tRNAPhe G34A |
||||||
|---|---|---|---|---|---|---|---|---|
| kcat, min−1 | Km, μM | kcat/Km, μM−1·min−1 | Relative, kcat/Km | kcat, min−1 | Km, μM | kcat/Km, μM−1·min−1 | Relative, kcat/Km | |
| WT | 2.3 ± 0.2 | 0.6 ± 0.1 | 4.1 ± 1.0 | 100 | ND | ND | 0.046 ± 0.005* | 1 |
| D400N | 2.6 ± 0.4 | 1.6 ± 0.4 | 1.6 ± 0.2 | 39 | 1.8 ± 0.1 | 4.1 ± 0.3 | 0.44 ± 0.04 | 11 |
| D400N/S411D | ND | ND | 0.43 ± 0.06* | 10 | ND | ND | 0.15 ± 0.002* | 4 |
| D400N/S411N | ND | ND | 0.24 ± 0.05* | 6 | ND | ND | 0.21 ± 0.02* | 5 |
| D400N/R450Q | ND | ND | 0.27 ± 0.03* | 7 | ND | ND | 0.39 ± 0.07* | 10 |
The concentrations of PheRS variants were normalized by active site titration. The kcat and Km values are for hmt-tRNAPhe. The kcat/Km is estimated by using subsaturating tRNA concentrations. ND, not determined.
*The Km values are too high to be accurately determined.