TABLE 2.
Kinetic parameters for cleavage of fIX and fIXα by fXIa determined from initial velocities
The initial velocities (v0) of cleavage after Arg145 in fIX, and cleavage after Arg180 in fIXα were plotted against initial substrate concentration, and analyzed using the Michaelis-Menten equation. Km and kcat were obtained from direct nonlinear least squares analysis using Scientist Software. Due to the low affinity between the enzyme and substrate, we were not able to reach saturation reactions with fXIa-CD, fXIa/PKA3, and fXIa-CD/PK-HC. Thus, values for Km are approximate and values for kcat were not determined.
| Protease | Substrate | Bond cleaved | kcat | Km | Catalytic efficiency |
|---|---|---|---|---|---|
| min−1 | μm | μm−1 min−1 | |||
| fXIa-WT | fIX | Arg145 | 10 ± 1 | 0.27 ± 0.07 | 40 ± 10 |
| fXIa-WT | fIXα | Arg180 | 7.1 ± 0.3 | 0.09 ± 0.01 | 80 ± 10 |
| fXIa-WT no Ca2+ | fIX | Arg145 | 4.6 ± 0.4 | 1.8 ± 0.3 | 3 ± 1 |
| fXIa-CD | fIX | Arg145 | 2.4 ± 0.4 | 3 ± 1 | 0.8 ± 0.3 |
| fXIa-CD | fIXα | Arg180 | NDa | >4 | 0.018 ± 0.001 |
| fXIa/PKA3 | fIX | Arg145 | 6 ± 1 | 3.9 ± 0.9 | 1.6 ± 0.4 |
| fXIa/PKA3 | fIXα | Arg180 | ND | >4 | 0.029 ± 0.003 |
| fXIa-CD/PK-HC | fIX | Arg145 | 5 ± 1 | 3 ± 1 | 2 ± 1 |
| fXIa-CD/PK-HC | fIXα | Arg180 | ND | >4 | 0.02 ± 0.01 |
a ND, not determined.