Functional properties of FIXa variants with substitutions in exosite II
| FIXa variant . | Amidolytic activity . | FX activation in the absence of FVIIIa . | FX activation in the presence of FVIIIa . | |||
|---|---|---|---|---|---|---|
| Apparent Km (mM) . | kcat (s−1) . | Apparent Km (nM) . | 103 × kcat (min−1) . | Apparent Km (nM) . | kcat (min−1) . | |
| wt-FIXa | 2.1 ± 0.3 | 13.2 ± 0.5 | 91 ± 6 | 25 ± 1 | 19 ± 6 | 54 ± 4 |
| FIXaK126ACT | 2.4 ± 0.3 | 10.9 ± 1.7 | 83 ± 19 | 15 ± 1 | 4 ± 1 | 26 ± 1 |
| FIXaR165ACT | 3.6 ± 0.5 | 11.1 ± 0.8 | 55 ± 9 | 7 ± 1 | ND | ND |
| FIXaR170ACT | 1.7 ± 0.2 | 11 ± 0.8 | 68 ± 15 | 17 ± 1 | 20 ± 6 | 100 ± 7 |
| FIXaK173ACT | 1.9 ± 0.2 | 9.3 ± 0.6 | 82 ± 12 | 11 ± 1 | 9 ± 5 | 39 ± 2 |
| FIXaK230ACT | 3.9 ± 0.7 | 13.3 ± 2.8 | 53 ± 13 | 14 ± 1 | 9 ± 2 | 30 ± 2 |
| FIXaR233ACT | 2.8 ± 0.3 | 11.4 ± 1.3 | 90 ± 24 | 14 ± 1 | 3 ± 1 | 17 ± 1 |
| FIXaR165A+R233ACT | 9.6 ± 3.3 | 14.2 ± 0.8 | 61 ± 14 | 5 ± 1 | ND | ND |
| FIXa variant . | Amidolytic activity . | FX activation in the absence of FVIIIa . | FX activation in the presence of FVIIIa . | |||
|---|---|---|---|---|---|---|
| Apparent Km (mM) . | kcat (s−1) . | Apparent Km (nM) . | 103 × kcat (min−1) . | Apparent Km (nM) . | kcat (min−1) . | |
| wt-FIXa | 2.1 ± 0.3 | 13.2 ± 0.5 | 91 ± 6 | 25 ± 1 | 19 ± 6 | 54 ± 4 |
| FIXaK126ACT | 2.4 ± 0.3 | 10.9 ± 1.7 | 83 ± 19 | 15 ± 1 | 4 ± 1 | 26 ± 1 |
| FIXaR165ACT | 3.6 ± 0.5 | 11.1 ± 0.8 | 55 ± 9 | 7 ± 1 | ND | ND |
| FIXaR170ACT | 1.7 ± 0.2 | 11 ± 0.8 | 68 ± 15 | 17 ± 1 | 20 ± 6 | 100 ± 7 |
| FIXaK173ACT | 1.9 ± 0.2 | 9.3 ± 0.6 | 82 ± 12 | 11 ± 1 | 9 ± 5 | 39 ± 2 |
| FIXaK230ACT | 3.9 ± 0.7 | 13.3 ± 2.8 | 53 ± 13 | 14 ± 1 | 9 ± 2 | 30 ± 2 |
| FIXaR233ACT | 2.8 ± 0.3 | 11.4 ± 1.3 | 90 ± 24 | 14 ± 1 | 3 ± 1 | 17 ± 1 |
| FIXaR165A+R233ACT | 9.6 ± 3.3 | 14.2 ± 0.8 | 61 ± 14 | 5 ± 1 | ND | ND |
Catalytic efficiency of FIXa variants of the exosite II was evaluated toward varying concentrations of the peptide substrate CH3SO2-(D)-CHG-Gly-Arg-pNA (indicated as amidolytic activity) and of the natural substrate FX in the presence of phospholipids and Ca2+ ions in the absence and presence of FVIIIa. Experimental conditions are given in “Materials and methods” and in the legend to Figure 5. Data were fitted in the Michaelis-Menten equation to obtain Km and kcat values. Kinetic parameters represent the mean ± standard deviation of 2 to 3 independent experiments.
ND, not determined because of substrate inhibition; see Figure 5C.