TABLE 4.
Enzyme kinetics of GalAK and GalK
GalAK activity was measured with varied concentrations of d-GalA (0.02–0.4 mm) and ATP (0.04–2.0 mm), after 5 min at standard conditions. GalK activity was measured with varied concentrations of d-Gal (0.04–8 mm) and ATP (0.04–8.0 mm), after 5 min at standard conditions. Enzyme velocities were plotted, and Solver software was used to generate best-fit curve and for calculation of Vmax and apparent Km. Each value is the mean of quadruple reactions, and the values varied by no more than ±10%. The kinetics of GalK obtained from H. sapiens, E. coli, S. cerevisiae, and P. furiosus ((40)), are displayed for comparison.
| Km(sugar) | Km (ATP) | Vmax (sugar) | Vmax (ATP) | kcat/Km | kcat/Km (ATP) | |
|---|---|---|---|---|---|---|
| μm | μm | μm s−1 μg−1 | μm s−1 μg−1 | s−1mm−1 | s−1mm−1 | |
| AtGalAK | 70.8 | 195 | 1.0 | 0.5 | 36 | 6.3 |
| AtGalK | 701 | 701 | 3.5 | 3.3 | 12.3 | 11.8 |
| HsGalK | 120 | 350 | 81.2 | 81.2 | 568 | 195 |
| EcGalK | 700 | 100 | 14 | 14 | 13.8 | 96.7 |
| ScGalK | 600 | 150 | 55.8 | 55.8 | 89.9 | 360 |
| PfGalK | 270 | 8 | 43.2 | 41.9 | 105 | 3439 |