TABLE 1.
Binding and kinetic constants for WT VahC and various active site mutants
Binding affinities and Michaelis constants were determined as described under “Experimental Procedures” using the fluorescence-quenching assay and the mART enzyme assay, respectively. The values represent the mean ± S.D. for at least triplicate independent measurements. Statistical analysis was performed using a one-way analysis of variance followed by Tukey's test. Differences in KD values among mutant and WT enzymes were not statistically significant (p > 0.05), and actin Km values were also not significantly different (p > 0.05). NAD+ Km values for the E215A, Y82A, and S178A mutants were statistically significant (p < 0.05).
| Mutant | KD | KM actin | KM NAD+ | kcat |
|---|---|---|---|---|
| μm | μm | μm | s−1 | |
| WT | 9 ± 0.4 | 24 ± 2 | 6 ± 0.4 | 22.3 ± 2.0 |
| E213A | 9 ± 0.7 | 20 ± 1 | 5 ± 0.4 | 0.11 ± 0.01 |
| E215A | 18 ± 3 | 23 ± 3 | 14 ± 2 | 0.05 ± 0.006 |
| Y77A | 11 ± 1 | 37 ± 5 | 8 ± 1 | 1.3 ± 0.09 |
| Y82A | 15 ± 2 | 35 ± 5 | 12 ± 0.5 | 0.65 ± 0.07 |
| N86A | 21 ± 2 | 11 ± 1 | 4 ± 0.1 | 9.35 ± 1.1 |
| S178A | 6 ± 0.3 | 33 ± 2 | 18 ± 1 | 1.38 ± 0.11 |