TABLE 2.
Steady-state kinetic parameters for HPG activation by equimolar complex of HPN·SK/SK random mutant(s) (RM)
Fixed, catalytic amounts of the respective preformed activator complexes of each protein with HPN were added to the cuvette containing HPG as the substrate and Chromozym®-PL in 50 mm Tris·Cl buffer, pH 7.5, and the reactions were monitored at 405 nm at 22 °C. The kinetic parameters for substrate HPG activation were determined as described under “Experimental Procedures.” NO, not observable.
| Activator protein (SK/SK*·HPN) | km | kcat | kcat/km |
|---|---|---|---|
| μm | min−1 | min−1/μm | |
| wtSK | 0.4 ± 0.05 | 7.5 ± 0.5 | 19.0 |
| RM 1 | NO | NO | NO |
| RM 2 | 0.5 ± 0.1 | 1.0 ± 0.2 | 2.0 |
| RM 3 | 0.5 ± 0.1 | 0.22 ± 0.03 | 0.4 |
| RM 4 | 0.5 ± 0.05 | 0.75 ± 0.05 | 1.5 |
| RM 5 | 0.6 ± 0.1 | 0.37 ± 0.03 | 0.6 |
| RM 6 | 0.6 ± 0.2 | 0.9 ± 0.02 | 1.5 |
| RM 7 | 0.5 ± 0.1 | 0.5 ± 0.02 | 1.0 |
| RM 8 | 0.6 ± 0.1 | 0.8 ± 0.05 | 1.3 |
| RM 9 | 0.6 ± 0.1 | 1.9 ± 0.1 | 3.1 |
| RM 10 | 1.2 ± 0.1 | 0.56 ± 0.05 | 0.5 |