Inhibitory activities of ZPI variants toward FXa and thermal stability of ZPI shutter region mutants
| . | Tm (°C) . | kapp (M−1s−1) . | SI . | kapp × SI(M−1s−1) . | SI (+PZ) . |
|---|---|---|---|---|---|
| ZPI | 58.5 ± 0.2 | 8.8 ± 2.1 × 103 | 2.1 ± 0.2 | 1.8 × 104 | 2.7 ± 0.2 |
| D213A | 58.4 ± 0.2 | 8.3 ± 2.4 × 103 | 1.9 ± 0.3 | 1.6 × 104 | 7.4 ± 0.2 |
| D213N | 68.5 ± 0.2 | 2.1 ± 0.7 × 103 | 4.6 ± 0.3 | 9.7 × 103 | 17.3 ± 0.2 |
| E231A | * | 3.7 ± 0.9 × 103 | 2.1 ± 0.3 | 7.8 × 103 | |
| D233A | 3.7 ± 1.4 × 103 | 1.9 ± 0.2 | 7.0 × 103 | ||
| E313A | 1.5 ± 0.3 × 103 | 4.3 ± 0.4 | 6.4 × 103 | ||
| K253A | 3.9 ± 0.5 × 103 | 1.7 ± 0.1 | 6.6 × 103 | ||
| K260A | 6.2 ± 1.6 × 103 | 1.7 ± 0.2 | 1.1 × 104 | ||
| K308/R310A | 3.6 ± 0.5 × 103 | 1.9 ± 0.4 | 6.8 × 103 |
| . | Tm (°C) . | kapp (M−1s−1) . | SI . | kapp × SI(M−1s−1) . | SI (+PZ) . |
|---|---|---|---|---|---|
| ZPI | 58.5 ± 0.2 | 8.8 ± 2.1 × 103 | 2.1 ± 0.2 | 1.8 × 104 | 2.7 ± 0.2 |
| D213A | 58.4 ± 0.2 | 8.3 ± 2.4 × 103 | 1.9 ± 0.3 | 1.6 × 104 | 7.4 ± 0.2 |
| D213N | 68.5 ± 0.2 | 2.1 ± 0.7 × 103 | 4.6 ± 0.3 | 9.7 × 103 | 17.3 ± 0.2 |
| E231A | * | 3.7 ± 0.9 × 103 | 2.1 ± 0.3 | 7.8 × 103 | |
| D233A | 3.7 ± 1.4 × 103 | 1.9 ± 0.2 | 7.0 × 103 | ||
| E313A | 1.5 ± 0.3 × 103 | 4.3 ± 0.4 | 6.4 × 103 | ||
| K253A | 3.9 ± 0.5 × 103 | 1.7 ± 0.1 | 6.6 × 103 | ||
| K260A | 6.2 ± 1.6 × 103 | 1.7 ± 0.2 | 1.1 × 104 | ||
| K308/R310A | 3.6 ± 0.5 × 103 | 1.9 ± 0.4 | 6.8 × 103 |
K308/R310A is a double mutant in which both K308 and R310 were replaced by Ala. In the presence of PZ, phospholipids, and Ca2+, these mutants also have similar ∼ 2- to 3-fold decreased inhibitory activity toward FXa compared with wild-type (data not shown). Tm of ZPI, second order inhibitory rate constant (kapp), and stoichiometry of ZPI-FXa reaction (SI) were measured, as described in “Methods.”
Missing values not determined.