TABLE 2.
Estimation of energy efficiencies in the presence of threonine and serine using wild type and mutant ThrRSs
Aminoacylation reactions were carried out at pH 8.0 and 37 °C. To determine the consumption of ATP and AA-tRNA, [α-32P]ATP and 14C-labeled amino acids were used in the parallel reactions, respectively. ThrRS wild type enzyme was used when threonine was the substrate and H73A as the post-transfer editing deficient mutants with serine. When the effect of aminoacyl transfer was investigated, the H73A/H309A double mutant was used. The progress curves were plotted and analyzed by linear regression. Values represent the mean ± S.E. from three experiments.
| Aminoacylation enzyme/AA substrate | Product monitored | Product formation rate | Ratio of ATP consumption to AA-tRNA formation |
|---|---|---|---|
| s−1 | |||
| Wild type ThrRS/Thr | [α-32P]AMP | 0.16 ± 0.01 | 1 |
| [14C]Thr-tRNAThr | 0.15 ± 0.01 | ||
| H73A ThrRS/Ser | [α-32P]AMP | 0.013 ± 0.0002 | 0.9 |
| [14C] Ser-tRNAThr | 0.015 ± 0.0003 | ||
| H73A/H309A ThrRS/Thr | [α-32P]AMP | 0.01 ± 0.0005 | 1 |
| [14C]Thr-tRNAThr | 0.01 ± 0.001 | ||
| H73A/H309A ThrRS/Ser | [α-32P]AMP | 0.002 ± 0.0002 | 2 |
| [14C]Ser-tRNAThr | 0.001 ± 0.0001 |