Table 2.
Kinetics of steady-state phenylalanylation of tRNA, and tRNAPhe enhancement of editing by wild-type E. coli PheRS and variants
| PheRS | CC503/X-gala | KM (L-Phe, μM) | kcat (min−1) | kcat/KM (min−1 μM−1) | k+tRNA/k−tRNAb | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Wild type | White | 10.3±1.0 | 103.3±3.7 | 10.0±0.1 | 1.5±0.1 | ||||||
| αA294G | White | 21.2±2.8 | 122.6±7.8 | 5.8±0.1 | 13.7±0.5 | ||||||
| αA294G, βH265A | Blue | 33.1±2.7 | 172.8±7.2 | 5.2±0.1 | 3.5±0.8 | ||||||
| αA294G, βH265L | Blue | 31.2±2.9 | 198.2±9.2 | 6.3±0.1 | 4.6±0.1 | ||||||
| αA294G, βE334A | Blue | 26.1±1.6 | 158.7±4.9 | 6.0±0.1 | 1.2±0.1 | ||||||
| αA294G, βT354W | Blue | 22.5±5.9 | 58.5±7.2 | 2.6±0.3 | 1.1±0.01 | ||||||
| αA294G, βA356W |
Blue |
28.2±1.3 |
187.0±4.4 |
6.6±0.1 |
1.3±0.1 |
||||||
| Color of colony upon expression of the corresponding PheRS-encoding gene in CC503-based indicator strains (see Figure 4B). | Fold enhancement of Tyr-dependent ATP hydrolysis upon addition of tRNA (see Table I). | ||||||||||