TABLE 3.
Steady state kinetic data
The initial rate of ATP hydrolysis was monitored using a pyruvate kinase and lactate dehydrogenase coupled assay. The kinetic constants kcat and Km were determined by plotting the initial rate versus the concentration of ATP and fitting the data to the Michaelis-Menten equation. The values are the average of at least two independent determinations, and the error is shown as S.D. ND, not determined.
| Protein | kcat | Km | kcat/Km |
|---|---|---|---|
| min−1 | mm | m−1 s−1 | |
| WT Nbp35 | 4.6 ± 0.1 | 0.4 ± 0.1 | 192 |
| WT Cfd1 | <0.7 | ND | ND |
| WT Nbp35-Cfd1 | 3.9 ± 0.2 | 5.0 ± 0.6 | 13 |
| K81ANbp35 | ND | >15 | ND |
| K86ANbp35 | 3.0 ± 0.1 | 0.6 ± 0.1 | 83 |
| D111ANbp35 | 1.1 ± 0.2 | 0.3 ± 0.1 | 61 |
| D111ANbp35-Cfd1 | 1.3 ± 0.1 | 6.0 ± 1.0 | 4 |
| Nbp35-D55ACfd1 | 4.7 ± 0.3 | 10.5 ± 1.0 | 7 |