TABLE 4.
Steady-state kinetic parameters for HPG activation by equimolar complexes of SK or the various deconvolved random mutants and HPN
The kinetic parameters for substrate HPG activation were determined at 22 °C with the chromogenic substrate in 50 mm Tris-Cl buffer, pH 7.5, 100 mm NaCl as described under “Experimental Procedures.” The data represent the mean of three independent determinations.
| Activator species | Km | kcat | kcat/Km |
|---|---|---|---|
| μm | min−1 | min−1μm−1 | |
| wt SK | 0.4 ± 0.05 | 7.5 ± 0.3 | 19.00 |
| R319H | 0.5 ± 0.02 | 0.17 ± 0.03 | 0.34 |
| R324G | 0.5 ± 0.01 | 7.0 ± 0.2 | 14.0 |
| D328A | 0.4 ± 0.03 | 1.7 ± 0.03 | 4.275 |
| D328E | 0.6 ± 0.04 | 3.2 ± 0.2 | 5.28 |
| D328G | 0.5 ± 0.01 | 0.75 ± 0.04 | 1.5 |
| D328N | 0.4 ± 0.03 | 2.2 ± 0.2 | 5.5 |
| D328R | 0.6 ± 0.02 | 0.84 ± 0.03 | 1.4 |
| P329L | 0.4 ± 0.03 | 7.0 ± 0.3 | 17.5 |
| R330H | 0.6 ± 0.06 | 0.68 ± 0.04 | 1.13 |
| K334A | 0.4 ± 0.02 | 3.42 ± 0.4 | 8.55 |
| K334E | 0.5 ± 0.06 | 0.9 ± 0.02 | 1.8 |
| L335Q | 0.7 ± 0.07 | 0.15 ± 0.01 | 0.2 |
| E355D | 0.5 ± 0.08 | 7.5 ± 0.2 | 15 |
| R363L | 0.5 ± 0.06 | 7.6 ± 0.4 | 15.2 |
| H381Q | 0.4 ± 0.04 | 7.7 ± 0.2 | 19.25 |