TABLE 3.
Steady-state kinetic parameters for amidase activity of equimolar complexes of SK or the various γ domain mutants and HPN
For the determination of the amidolytic parameters, wt SK/SK mutants were precomplexed with HPN in an equimolar ratio, and an aliquot of this mixture was assayed for amidolysis at varying concentrations of two chromogenic substrates with different peptide sequences, namely chromozym-PL(tosyl-Gly-l-Pro-l-Lys-pNA) and S-2444 (l-PyroGlu-Gly-l-Arg-pNA) as described under “Experimental Procedures.” The data represent the mean of three independent determinations.
| Activator specie | Chromozym-PL |
S-2444 |
||||
|---|---|---|---|---|---|---|
| Km | kcat | kcat/Km | Km | kcat | kcat/Km | |
| mm | min−1 | min−1/mm | mm | min−1 | min−1/mm | |
| HPN | 0.2 ± 0.06 | 32 ± 2 | 160 | 1.0 ± 0.1 | 8.0 ± 0.4 | 8.0 |
| wtSK·HPN | 0.5 ± 0.05 | 30 ± 2 | 60 | 3.1 ± 0.2 | 6.0 ± 0.4 | 1.9 |
| SK-D328A·HPN | 0.6 ± 0.03 | 29 ± 1 | 48 | 3.2 ± 0.4 | 6.5 ± 0.2 | 2.0 |
| SK-K334E·HPN | 0.5 ± 0.05 | 30 ± 1.5 | 60 | 2.8 ± 0.2 | 6.0 ± 0.3 | 2.1 |
| SK-G344D·HPN | 0.6 ± 0.04 | 31 ± 3 | 51 | 3.5 ± 0.3 | 6.2 ± 0.1 | 1.7 |