Table 2.
Kinetic Constants for hydrolysis of FXIII (28–41) V34, V34L, V34P, and V34F Activation Peptides with different IIa mutantsa
| FXIII (28–41) V34 AP | Km (μM) | kcat (s−1) | kcat/Km (μM−1.s−1) |
|---|---|---|---|
| WT IIab | 298 ± 42 | 2.57 ±0.005 | 8.6 × 10−3 ± 1.0 × 10−3 |
| W60dA IIa | 1375 ± 422 | 0.86 ±0.046 | 6.3 × 10−4 ± 1.9 × 10−4 |
| Y60aA IIa | 678 ±120 | 0.048 ± 0.0032 | 7.1 × 10−5 ± 1.3 × 10−5 |
| L99A IIab | 2074 ± 414 | 0.89 ± 0.073 | 4.3 × 10−4 ± 1.0 × 10−4 |
| W215A IIab | 2374 ± 372 | 5.53 ± 0.017 | 2.3 × 10−3 ± 4.0 × 10−4 |
| E217A IIab | 1242 ± 392 | 2.84 ± 0.014 | 2.3 × 10−3 ± 7.0 × 10−4 |
| WE (W215A/E217A) IIab | 745 ± 98 | 0.117 ± 0.008 | 1.5 × 10−4 ± 2.0 × 10−5 |
| FXIII (28–41) V34L AP | Km (μM) | kcat (s−1) | kcat/Km (μM−1.s−1) |
|---|---|---|---|
| WT IIab | 315 ± 42 | 22.9 ± 0.003 | 7.3 × 10−2 ± 1.2 × 10−2 |
| W60dA IIa | 1000 ± 201 | 5.07 ± 0.021 | 5.1 × 10−3 ± 1.0 × 10−3 |
| Y60aA IIa | 696 ± 161 | 0.37 ± 0.012 | 5.3 × 10−4 ± 1.2 × 10−4 |
| L99A IIab | 1930 ± 354 | 5.94 ± 0.02 | 3.1 × 10−3 ± 2.0 × 10−4 |
| W215A IIab | 3619 ± 470 | 5.31 ± 0.002 | 1.5 × 10−3 ± 2.0 × 10−4 |
| E217A IIab | 1025 ± 121 | 9.93 ± 0.02 | 9.7 × 10−3 ± 1.0 × 10−3 |
| WE (W215A/E217A) IIab | 142 ± 28 | 0.052 ± 0.003 | 3.6 × 10−4 ± 1.0 × 10−4 |
| FXIII (28–41) V34P AP | Km (μM) | kcat (s−1) | kcat/Km (μM−1.s−1) |
|---|---|---|---|
| WT IIa | 133 ± 10 | 2.17 ± 0.0021 | 1.6 × 10−2 ± 1.3 × 10−3 |
| W60dA IIa | 605 ± 97 | 1.61 ± 0.127 | 2.7 × 10−3 ± 7.9 × 10−4 |
| Y60aA IIa | 810 ± 144 | 0.085 ± 0.002 | 1.0 × 10−4 ± 2.0 × 10−5 |
| L99A IIa | 749 ± 118 | 0.111 ± 0.010 | 2.0 × 10−4 ± 3.0 × 10−5 |
| W215A IIa | 2662 ± 193 | 0.947 ± 0.0365 | 3.6 × 10−4 ± 3.0 × 10−5 |
| E217A IIa | 947 ± 93 | 1.69 ± 0.079 | 1.8 × 10−3 ± 1.9 × 10−4 |
| WE (W215A/E217A) IIa | N/A | N/A | N/A |
| FXIII (28–41) V34F AP | Km (μM) | kcat (s−1) | kcat/Km (μM−1.s−1) |
|---|---|---|---|
| WT IIa | 442 ± 93 | 6.27 ± 0.54 | 1.4 × 10−2 ± 3.0 × 10−3 |
| W60dA IIa | 435 ± 98 | 1.46 ± 0.15 | 3.4 × 10−3 ± 8.0 × 10−4 |
| Y60aA IIa | N/A | N/A | N/A |
| L99A IIa | 570 ± 125 | 2.73 ± 0.3 | 5.0 × 10−3 ± 1.0 × 10−3 |
| W215A IIa | 437 ± 81 | 1.97 ± 0.14 | 4.5 × 10−3 ± 9.0 × 10−4 |
| E217A IIa | 454 ± 102 | 3.36 ± 0.34 | 7.4 × 10−3 ± 2.0 × 10−3 |
| WE (W215A/E217A) IIa | 190 ± 43 | 0.067 ± 0.005 | 3.5 × 10−4 ± 8.0 × 10−5 |
a
Kinetic constants for the thrombin-catalyzed hydrolysis reactions were determined from an HPLC assay as described in Materials and Methods. The results shown here represent averages of at least three independent experiments. Kinetic values were calculated using nonlinear regression analysis methods using SigmaPlot. The error values correspond to standard error of the mean (SEM).
b
Isetti and Maurer [37].