Table 1.
Steady-state kinetic parameters in formylation using wild-type and mutant MTFs and wild-type and mutant initiator tRNAs
| tRNA used | Enzyme | Km, μM | kcat, s−1 | kcat/Km, μM−1⋅s−1 | Relative ratio |
|---|---|---|---|---|---|
| Wild type | WT | 0.56 ± 0.14 | 41.52 ± 13.85 | 72.48 | 1.00 |
| G41R | 0.66 ± 0.16 | 11.40 ± 0.159 | 18.49 | 0.25 | |
| G41K | 1.02 ± 0.03 | 14.60 ± 0.053 | 14.28 | 0.20 | |
| U35A36 | WT | 2.90 ± 1.35 | 8.85 ± 2.29 | 3.13 | 1.00 |
| G41R | 2.30 ± 1.12 | 2.78 ± 0.94 | 1.21 | 0.38 | |
| G41K | 2.01 ± 0.50 | 2.95 ± 0.56 | 1.64 | 0.52 | |
| U35A36/G72G73 | WT | 5.70 ± 0.89 | (1.48 ± 0.41) × 10−3 | 2.67 × 10−4 | 1.00 |
| G41R | 5.27 ± 1.18 | (3.45 ± 0.71) × 10−2 | 6.93 × 10−3 | 25.95 | |
| G41K | 5.19 ± 0.07 | (3.71 ± 0.28) × 10−2 | 7.14 × 10−3 | 26.69 |
Kinetic parameters were measured by using Lineweaver–Burk and Eadie–Hofstee or Hanes–Woolf plots. These gave basically the same numbers. The kinetic parameters listed are the average of four to six separate measurements. The tRNA concentrations used were 0.2–2 μM for wild-type (WT) tRNA and 0.5–6 μM for the U35A36 and the U35A36/G72G73 mutant tRNAs. The enzyme concentrations used varied from 0.025 to 0.106 nM for the wild-type tRNA, 0.34 to 0.65 nM for the U35A36 tRNA, and 0.12 to 1.55 μM for the U35A36/G72G73 tRNA. For calculation of relative ratio of kcat/Km, the kcat/Km of wild-type MTF for each tRNA is taken as 1.