TABLE 1.
Steady-state kinetics of amino acid activation by E. coli wild-type PheRS and two active site PheRS mutants
| PheRS | Phe |
m-Tyr |
Tyra |
Specificity |
|||||
|---|---|---|---|---|---|---|---|---|---|
| Km (μM) | kcat (s−1) | kcat/Km (s−1/μM) | Km (μM) | kcat (s−1) | kcat/Km (s−1/μM) | kcat/Km (s−1/μM) | Phe/m-Tyr | Phe/Tyr | |
| WT | 21 ± 1 | 61 ± 11 | 2.9 | 175 ± 31 | 23 ± 3 | 0.13 | 0.002 ± 0.0004 | 22 | 1,400 |
| αA294G | 59 ± 29 | 58 ± 16 | 1.0 | 62 ± 7 | 36 ± 6 | 0.6 | 0.01 ± 0.006 | 1.8 | 110 |
| αA294S | 27 ± 7 | 97 ± 10 | 3.6 | NDa | NDa | 0.01 ± 0.003 | 0.001 ± 0.0002 | 370 | 3,700 |
a
Individual kinetic parameters could not be determined due to a high Km and substrate solubility. kcat/Km was estimated by ν = kcat/Km ([E][S]). Standard deviation is from 3 replicates. ND, not determined.