TABLE 3.
Parallel formation of AMP and aa-tRNA by SgIleRS and ScIleRS and their deacylation variants
The values represent the best fit value ± S.E. of three independent experiments.
| k(AMP)/s−1 | k(aa-tRNA)/s−1 | k(AMP)/k(aa-tRNA) | ktRNA-ind/s−1 | |
|---|---|---|---|---|
| SgIleRS WT + Ilea | 0.64 ± 0.08 | 0.65 ± 0.05 | 0.98 | NDb |
| SgIleRS WT + Vala | 0.83 ± 0.2 | 0.13 ± 0.02 | 6.24 | 0.004 |
| SgIleRS D334A + Ilea | 0.20 ± 0.02 | 0.19 ± 0.02 | 1.05 | ND |
| SgIleRS D334A + Vala | 0.20 ± 0.04 | 0.19 ± 0.04 | 1.05 | 0.004 |
| ScIleRS WT + Ile | 0.20 ± 0.03 | 0.13 ± 0.02 | 1.60 | ND |
| ScIleRS WT + Val | 0.53 ± 0.08 | 0.023 ± 0.007 | 23 | 0.002 |
| ScIleRS D333A + Ile | 0.038 ± 0.006 | 0.030 ± 0.008 | 1.27 | ND |
| ScIleRS D333A + Val | 0.09 ± 0.01 | 0.041 ± 0.012 | 2.19c | 0.002 |
a The reactions with SgIleRS were measured in the presence of EF-Tu. In the absence of EF-Tu, aa-tRNA accumulated at lower concentrations (down to 10% of the product), depending on the IleRS concentration added.
b ND means not determined due to insufficient activity.
c A portion of Val-AMP accumulates in solution with rate k(Val-AMP) = 0.021 ± 0.004 s−1 participating in the selective release pathway. If both Val-AMP and AMP are taken into account, the ratio of used ATP per aa-tRNA is raised to 2.6.