Kinetic constants for the cleavage of Spectrozyme PCa and inhibition by p-aminobenzamidine (PAB) for wild-type and mutant enzymes
. | Km, μM . | kcat, S−1 . | kcat/Km, s−1 μM−1 . | Ki (PAB), μM . |
|---|---|---|---|---|
| APC | 134.2 ± 5.3 | 33.2 ± 0.4 | 0.25 ± 0.01 | 40.2 ± 2.7 |
| APC-fXa143-154 | 92.7 ± 6.1 | 56.9 ± 1.0 | 0.61 ± 0.05 | 19.3 ± 1.1 |
| Thrombin | 6.5 ± 1.5 | 80.3 ± 5.0 | 12.4 ± 3.6 | 51.6 ± 2.7 |
| Thrombin-fXa143-154 | 7.5 ± 0.8 | 82.2 ± 2.2 | 11.0 ± 1.5 | 51.9 ± 2.8 |
. | Km, μM . | kcat, S−1 . | kcat/Km, s−1 μM−1 . | Ki (PAB), μM . |
|---|---|---|---|---|
| APC | 134.2 ± 5.3 | 33.2 ± 0.4 | 0.25 ± 0.01 | 40.2 ± 2.7 |
| APC-fXa143-154 | 92.7 ± 6.1 | 56.9 ± 1.0 | 0.61 ± 0.05 | 19.3 ± 1.1 |
| Thrombin | 6.5 ± 1.5 | 80.3 ± 5.0 | 12.4 ± 3.6 | 51.6 ± 2.7 |
| Thrombin-fXa143-154 | 7.5 ± 0.8 | 82.2 ± 2.2 | 11.0 ± 1.5 | 51.9 ± 2.8 |
The kinetic constants were calculated from the cleavage rate of increasing concentrations of SpPCa (0.75-2000 μM) by wild-type and mutant APC (2 nM) and wild-type and mutant thrombin (0.5 nM) in TBS/Ca2+ as described in “Materials and methods.” The kinetic values are averages of 3 measurements ± standard errors. The Ki values for PAB were measured from the decrease in initial rates of S2266 hydrolysis produced by the competitive inhibitor in TBS/Ca2+. The global fitting of data to a competitive binding equation yielded values for Ki (last column) and Km and kcat values for the hydrolysis of S2266 (146.3 ± 8.2 μM and 8.9 ± 0.1 s−1 for APC; 114.4 ± 5.7 μM and 19.0 ± 0.2 s−1 for APC-fXa143-154; 146.5 ± 6.1 μM and 12.8 ± 0.2 s−1 for thrombin; and 209.8 ± 9.5 μM and 12.3 ± 0.2 s−1 for thrombin-fXa143-154).