Kinetic constants for macromolecular substrate cleavage
| . | Enzyme/substrate . | |||||||
|---|---|---|---|---|---|---|---|---|
| Prothrombinase*/prothrombin . | FXa-FVa/prothrombin . | Prothrombinase/prethrombin-1 . | FXa FVa/prethrombin-1 . | |||||
| Km, μM . | kcat, min−1 . | Km, μM . | kcat, min−1 . | Km, μM . | kcat, min−1 . | Km, μM . | kcat, min−1 . | |
| pt-rFV | 0.39 ± 0.04 | 1250 ± 38 | 1.83 ± 0.12 | 352 ± 11 | 1.12 ± 0.08 | 1610 ± 28 | 1.09 ± 0.11 | 1570 ± 38 |
| pt-rFV-QQ | 0.44 ± 0.05 | 1270 ± 39 | 1.87 ± 0.05 | 334 ± 4 | 1.17 ± 0.06 | 1670 ± 24 | 1.20 ± 0.11 | 1560 ± 34 |
| pt-rFVa | 0.41 ± 0.03 | 1350 ± 31 | 1.74 ± 0.15 | 347 ± 1 | 1.38 ± 0.06 | 2210 ± 26 | 1.38 ± 0.06 | 2170 ± 43 |
| hFV-810 | 0.48 ± 0.07 | 1400 ± 62 | NA | NA | ND | ND | ND | ND |
| . | Enzyme/substrate . | |||||||
|---|---|---|---|---|---|---|---|---|
| Prothrombinase*/prothrombin . | FXa-FVa/prothrombin . | Prothrombinase/prethrombin-1 . | FXa FVa/prethrombin-1 . | |||||
| Km, μM . | kcat, min−1 . | Km, μM . | kcat, min−1 . | Km, μM . | kcat, min−1 . | Km, μM . | kcat, min−1 . | |
| pt-rFV | 0.39 ± 0.04 | 1250 ± 38 | 1.83 ± 0.12 | 352 ± 11 | 1.12 ± 0.08 | 1610 ± 28 | 1.09 ± 0.11 | 1570 ± 38 |
| pt-rFV-QQ | 0.44 ± 0.05 | 1270 ± 39 | 1.87 ± 0.05 | 334 ± 4 | 1.17 ± 0.06 | 1670 ± 24 | 1.20 ± 0.11 | 1560 ± 34 |
| pt-rFVa | 0.41 ± 0.03 | 1350 ± 31 | 1.74 ± 0.15 | 347 ± 1 | 1.38 ± 0.06 | 2210 ± 26 | 1.38 ± 0.06 | 2170 ± 43 |
| hFV-810 | 0.48 ± 0.07 | 1400 ± 62 | NA | NA | ND | ND | ND | ND |
The data are representative of 2 independent measurements. The errors in the fitted constants represent ± 2 SD.
ND indicates not determined. NA, for this experiment, rates were very low precluding an accurate assessment of kinetic parameters; however, we estimate the Km to be approximately 3 μM. The kcat value is difficult to estimate because the Kd between snake FXa and hFV-810 in solution is very high (Table 2); thus, the total enzyme concentration in this experiment is not known with certainty.
Prothrombinase is defined as FXa and FVa bound to a negatively charged membrane in the presence of calcium.