Kinetic constants and stoichiometries of inhibition for ZPI-factor Xa reactions in the absence and presence of cofactors and KDs for ZPI-PZ interactions
| ZPI . | No cofactors . | Cofactors . | |||||
|---|---|---|---|---|---|---|---|
| kass,uncat, M−1s−1 . | SI, mol I/mol E . | kass,uncat × SI, M−1s−1 . | kass,cat, M−1s−1 . | SI, mol I/mol E . | kass,cat × SI, M−1s−1* . | KD ZPI-PZ, M . | |
| Wild-type | 1.0 ± 0.1 × 104 | 3.6 ± 0.4 | 3.6 ± 0.8 × 104 | 3.1 ± 0.1 × 106 | 2.8 ± 0.2 | 8.7 ± 0.9 × 106 | 1.2 ± 0.1 × 10−9 |
| M71A | 8.6 ± 0.7 × 103 | 3.7 ± 0.1 | 3.2 ± 0.3 × 104 | 2.8 ± 0.3 × 106 | 2.9 ± 0.1 | 8.1 ± 1.1 × 106 | 1.7 ± 0.5 × 10−8 |
| D74A | 9.5 ± 0.1 × 103 | 4.1 ± 0.1 | 3.9 ± 0.1 × 104 | 3.3 ± 0.2 × 106 | 2.6 ± 0.1 | 8.6 ± 0.9 × 106 | 3.4 ± 0.5 × 10−8 |
| D238A | 6.0 ± 0.4 × 103 | 5.5 ± 0.4 | 3.3 ± 0.5 × 104 | 3.1 ± 0.1 × 106 | 2.9 ± 0.1 | 9.0 ± 0.9 × 106 | ≤ 1 × 10−9 |
| K239A | 9.3 ± 0.2 × 103 | 4.0 ± 0.1 | 3.7 ± 0.2 × 104 | 2.4 ± 0.1 × 106 | 4.2 ± 0.1 | 10 ± 1 × 106 | ≤ 1 × 10−9 |
| Y240A | 5.8 ± 0.1 × 103 | 4.7 ± 0.1 | 2.7 ± 0.1 × 104 | ND | ND | 1-1.5 × 10−6 | |
| D293A | 7.6 ± 0.6 × 103 | 3.8 ± 0.1 | 2.9 ± 0.3 × 104 | ND | ND | 4.0 ± 1.4 × 10−5 | |
| D74A/D293A | 1.1 ± 0.1 × 104 | 4.7 ± 0.1 | 5.2 ± 0.6 × 104 | ND | ND | 2.4 ± 1.6 × 10−4 | |
| ZPI . | No cofactors . | Cofactors . | |||||
|---|---|---|---|---|---|---|---|
| kass,uncat, M−1s−1 . | SI, mol I/mol E . | kass,uncat × SI, M−1s−1 . | kass,cat, M−1s−1 . | SI, mol I/mol E . | kass,cat × SI, M−1s−1* . | KD ZPI-PZ, M . | |
| Wild-type | 1.0 ± 0.1 × 104 | 3.6 ± 0.4 | 3.6 ± 0.8 × 104 | 3.1 ± 0.1 × 106 | 2.8 ± 0.2 | 8.7 ± 0.9 × 106 | 1.2 ± 0.1 × 10−9 |
| M71A | 8.6 ± 0.7 × 103 | 3.7 ± 0.1 | 3.2 ± 0.3 × 104 | 2.8 ± 0.3 × 106 | 2.9 ± 0.1 | 8.1 ± 1.1 × 106 | 1.7 ± 0.5 × 10−8 |
| D74A | 9.5 ± 0.1 × 103 | 4.1 ± 0.1 | 3.9 ± 0.1 × 104 | 3.3 ± 0.2 × 106 | 2.6 ± 0.1 | 8.6 ± 0.9 × 106 | 3.4 ± 0.5 × 10−8 |
| D238A | 6.0 ± 0.4 × 103 | 5.5 ± 0.4 | 3.3 ± 0.5 × 104 | 3.1 ± 0.1 × 106 | 2.9 ± 0.1 | 9.0 ± 0.9 × 106 | ≤ 1 × 10−9 |
| K239A | 9.3 ± 0.2 × 103 | 4.0 ± 0.1 | 3.7 ± 0.2 × 104 | 2.4 ± 0.1 × 106 | 4.2 ± 0.1 | 10 ± 1 × 106 | ≤ 1 × 10−9 |
| Y240A | 5.8 ± 0.1 × 103 | 4.7 ± 0.1 | 2.7 ± 0.1 × 104 | ND | ND | 1-1.5 × 10−6 | |
| D293A | 7.6 ± 0.6 × 103 | 3.8 ± 0.1 | 2.9 ± 0.3 × 104 | ND | ND | 4.0 ± 1.4 × 10−5 | |
| D74A/D293A | 1.1 ± 0.1 × 104 | 4.7 ± 0.1 | 5.2 ± 0.6 × 104 | ND | ND | 2.4 ± 1.6 × 10−4 | |
Rate constants in the absence of cofactors (kass,uncat) were obtained from the slope of linear plots of pseudo-first-order rate constants (kobs) on ZPI concentration in the range of 100-800nM. Rate constants for reactions in the presence of cofactors (kass,cat) were obtained from the data shown in Figure 4. kass,cat could not be determined for ZPI variants with low PZ affinity (Y240A, D293A, and D74A/D293A). Stoichiometry of inhibition (SI) values were determined from stoichiometric titrations of factor Xa with ZPI as described in “Stoichiometry of ZPI-protease reactions.” The product of kass,uncat or kass,cat and SI represents the corrected association rate constant for reaction through the inhibitory pathway. KDs for ZPI-PZ interactions were determined from fits of the kinetic titrations shown in Figures 4 and 6 using the equation provided in “Kinetics of ZPI-FXa reactions.”
ND indicates not determined.
Wild-type rate constants are somewhat lower than those measured in previous studies9,11 because of the reduced calcium concentration.