Table 1.
Comparative kinetic properties of HsaDRS2 and EcoDRS in tRNA aspartylation
| DRSa | tRNAAsp (transcript) | kcat (10−3 sec−1) | KM (µM) | kcat/KM (10−3 sec−1 µM−1) | L | ΔΔGb (kcal mol−1) |
|---|---|---|---|---|---|---|
| HsaDRS2 | mt | 4.0 ± 0.4 | 1.1 ± 0.2 | 3.7 ± 0.7 | 72 | −2.45 |
| HsaDRS2 | Eco | 10.3 ± 2.0 | 1.2 ± 0.1 | 8.3 ± 0.8 | 35 | −2.04 |
| EcoDRS | Eco | 230 ± 6.0 | 0.8 ± 0.1 | 293 ± 14.4 | 1 | – |
| EcoDRS | mt | nd | nd | nd | >106 |
All data were obtained at 25°C (T = 298°K) and correspond to mean values from at least duplicated experiments with independent and freshly prepared enzyme and tRNA lots.
akcat and KM values were derived from assays containing 2 nM of EcoDRS or 50 nM of HsaDRS2 and 0.2–4 µM of Eco or Hsa mt-tRNA transcript. L stands for the relative loss in aminoacylation efficiency referred to the tRNA aminoacylation efficiency of EcoDRS for Eco tRNAAsp [L = (kcat/KM)Eco system/(kcat/KM)other]; bΔΔG is the variation of the free energy change at the transition state when comparing aminoacylation in the homologous Eco system with the reactions in the heterologous or Hsa mt-systems [ΔΔG = −RT lnL] (37); nd for not detectable.