Table 5.
Effect of MnmE substitutions on GTPase cycle and tRNA modification status
| MnmE protein | GTP binding k1 (min−1) | G-domain dimerization k2 (min−1) | GTP hydrolysis k3 (min−1) | G-domain dissociation k4 (min−1) | Pi release k5 (min−1) | tRNA-modification activity (%) |
|---|---|---|---|---|---|---|
| wt | 3528 | 717 | 201 | 12.4 ± 0.6 | 9.6 ± 1.0 | 100 |
| Fast hydrolase activity variants | ||||||
| T250S | 3452 | 356 | 163 | 2.6 ± 0.3 | 2.7 ± 0.3 | 19 |
| R256A | 2633 | 207 | 44 | 5.1 ± 0.8 | 5.5 ± 0.6 | 97 |
| L274G | 2761 | 418 | 30 | 11.1 ± 0.6 | 1.6 ± 0.1 | 0 |
| L274A | 3222 | 378 | 56 | 12.0 ± 0.8 | 1.5 ± 0.2 | 0 |
| G285A | 3196 | 280 | 42 | 10.3 ± 0.5 | 0.8 ± 0.1 | 0 |
| Slow hydrolase activity variants | ||||||
| T251A | 2743 | 236 | 0.01 | 0 | ||
| R252A | 3935 | 755 | 6.74 | 1.3 ± 0.1 | 0.8 ± 0.1 | 5 |
| D253A | 3389 | 266 | 5.71 | 1.7 ± 0.2 | 0.3 ± 0.1 | 0 |
| L274Q | 3254 | 222 | 2.13 | 1.9 ± 0.3 | 2.4 ± 0.2 | 19 |
| R275A | 3216 | 218 | 12.43 | 1.9 ± 0.2 | 2.0 ± 0.2 | 51 |
| E282A | 2241 | 104 | 0.02 | 0 | ||
| G285I | 2323 | 151 | 0.33 | 0 | ||
| R288A | 3314 | 341 | 8.22 | 2.3 ± 0.3 | 0.9 ± 0.1 | 11 |
Kinetic constants from single-turnover assays were determined from data as those shown in Figure 7. Results are mean ± SD of at least three independent experiments. When not indicated, standard deviations of rate constants were around ±10%, except for T251A and G285I in which the standard deviation of k3 was around ±30%. Data on tRNA modification activity of MnmE proteins were taken from Table 4. Bold numbers are used to highlight the implication of the corresponding MnmE substitution in G-domain dimerization, GTP hydrolysis, G-domain dissociation and/or Pi release.