TABLE 1.
Kinetic parameters for cleavage of fIX and fIXα to fIXaβ by fXIa
Fitting of full-progress experimental traces was performed with KinTek software (KinTek Explorer version 2.5) using the reaction equations shown under “Experimental Procedures.” Km and kcat for activation were calculated from individual rate constants for each step. Ki values were fixed to lower limits obtained by surface plasmon resonance. Values are the mean ± S.D. for each experiment.
| Protease | Substrate | kcat | Kd | Km | Catalytic efficiency | Ki |
|---|---|---|---|---|---|---|
| min−1 | μm | μm−1 min−1 | μm | |||
| Cleavage of fIX after Arg145 | ||||||
| fXIa-WT | fIX | 12 ± 1 | 0.10 ± 0.01 | 0.20 ± 0.01 | 60 ± 6 | 0.14 |
| fXIa-WT no Ca2+ | fIX | 6.5 ± 0.4 | 4.9 ± 0.4 | 4.9 ± 0.4 | 1.3 ± 0.3 | 5 |
| fXIa-CD | fIX | 3.2 ± 0.1 | 4.9 ± 0.2 | 4.9 ± 0.2 | 0.7 ± 0.1 | 3 |
| fXIa/PKA3 | fIX | 4.9 ± 0.1 | 4.9 ± 0.2 | 4.9 ± 0.2 | 1.0 ± 0.1 | 3 |
| fXIa/PKA2-Ser140 | fIX | 16 ± 1 | 0.03 ± 0.01 | 0.10 ± 0.01 | 160 ± 20 | 0.15 |
| fXIa-CD/PK-HC | fIX | 1.6 ± 0.1 | 2.4 ± 0.2 | 2.4 ± 0.2 | 0.7 ± 0.1 | 5 |
| Cleavage of fIX after Arg180a | ||||||
| fXIa-WT | fIX | 35 ± 6 | 0.06 ± 0.01 | 0.08 ± 0.01 | 400 ± 100 | 0.06 |
| fXIa-WT | fIXα | 12 ± 1 | 0.08 ± 0.01 | 0.09 ± 0.01 | 130 ± 20 | 0.06 |
| fXIa-WT no Ca2+ | fIX | 3.6 ± 0.2 | 15 ± 1 | 15 ± 1 | 0.24 ± 0.04 | 5 |
| fXIa-CD | fIX | 0.5 ± 0.1 | 11 ± 1 | 11 ± 1 | 0.05 ± 0.01 | 5 |
| fXIa-CD | fIXα | 0.5 ± 0.1 | 13 ± 2 | 13 ± 2 | 0.04 ± 0.01 | 5 |
| fXIa/PKA3 | fIX | 0.6 ± 0.1 | 7 ± 1 | 7 ± 1 | 0.09 ± 0.02 | 3 |
| fXIa/PKA3 | fIXα | 0.6 ± 0.1 | 7 ± 1 | 7 ± 1 | 0.09 ± 0.02 | 3 |
| fXIa/PKA2-Ser140 | fIX | 40 ± 10 | 0.04 ± 0.01 | 0.08 ± 0.02 | 500 ± 200 | 0.06 |
| fXIa-CD/PK-HC | fIX | 0.4 ± 0.1 | 10 ± 1 | 10 ± 1 | 0.04 ± 0.01 | 5 |
| fXIa-CD/PK-HC | fIXα | 0.4 ± 0.1 | 10 ± 1 | 10 ± 1 | 0.04 ± 0.01 | 5 |
a Due to the low affinity between the enzyme and substrate in certain reactions, we were not able to reach saturation for reactions with fXIa-CD, fXIa/PKA3, fXIa-CD/PK-HC, or fXIa-WT in the absence of Ca2+. Fits for kcat and Km are linked, with the listed Km values representing lower estimates for the reaction. kcat/Km is the appropriate parameter for comparing these reactions.