TABLE 2.
Constants for the binding of active site-directed fluorescent probes to meizothrombin variants
Constants ± 95% confidence limits were determined from global fitting according to Scheme 2 (mIIa-ΔF1) or Scheme 3 (all other mIIa variants). Symbolic constants correspond to those listed in the two schemes.
| Species | Probe | k+1 | k−1 | fea | fE | k+2 | k−2 | KE,Pb | k+3 | k−3 | Ke,P | KConf |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| s−1 | s−1 | μm−1.s−1 | s−1 | nm | μm−1.s−1 | s−1 | nm | |||||
| mIIa-ΔF1 | I-2581 | 43.3 ± 0.9 | 3.10 ± 0.3 | 0.07 ± 0.01 | 0.93 ± 0.09 | 49.7 ± 1.1 | 1.2 ± 0.01 | 24.1 ± 0.2 | NAc | NA | NA | NA |
| mIIa-ΔF1 | DAPA | 26.7 ± 0.3 | 5.25 ± 0.4 | 0.16 ± 0.01 | 0.84 ± 0.06 | 90.3 ± 1.1 | 0.44 ± 0.01 | 4.9 ± 0.1 | NA | NA | NA | NA |
| mIIaQQQd | I-2581 | 1.67 ± 0.01 | 1.93 ± 0.02 | 0.54 | 0.46 | 57.3 ± 0.2 | 0.54 ± 0.01 | 9.3 ± 0.01 | 1.76 ± 0.01 | 2.30 ± 0.23 | 1310 ± 110 | 0.008 ± 0.001 |
| mIIaQQQd | DAPA | 1.70 ± 0.17 | 1.89 ± 0.08 | 0.53 | 0.47 | 92.5 ± 0.6 | 0.26 ± 0.01 | 2.84 ± 0.05 | 2.33 ± 0.07 | 0.58 ± 0.06 | 250 ± 20 | 0.013 ± 0.003 |
| dG-mIIaQQQ | I-2581 | 2.76 ± 0.03 | 1.87 ± 0.02 | 0.40 ± 0.01 | 0.60 ± 0.01 | 24.6 ± 0.2 | 0.33 ± 0.01 | 13.4 ± 0.4 | 1.59 ± 0.27 | 3.94 ± 0.44 | 2480 ± 350 | 0.004 ± 0.001 |
| dG-mIIaQQQ | DAPA | 1.95 ± 0.02 | 1.70 ± 0.02 | 0.47 ± 0.01 | 0.53 ± 0.01 | 41.5 ± 0.2 | 0.20 ± 0.01 | 4.8 ± 0.24 | 1.56 ± 0.02 | 1.07 ± 0.17 | 686 ± 109 | 0.006 ± 0.001 |
| dG-mIIaA195 | I-2581 | 1.59 ± 0.10 | 1.71 ± 0.06 | 0.52 ± 0.02 | 0.48 ± 0.02 | 30.0 ± 0.2 | 0.24 ± 0.03 | 8.0 ± 1.0 | 1.76 ± 0.03 | 1.49 ± 0.19 | 847 ± 108 | 0.010 ± 0.001 |
| dG-mIIaA195 | DAPA | 1.42 ± 0.10 | 1.95 ± 0.05 | 0.58 ± 0.02 | 0.42 ± 0.01 | 33.8 ± 0.4 | 0.26 ± 0.02 | 7.7 ± 0.6 | 3.22 ± 0.05 | 3.78 ± 0.23 | 1174 ± 72 | 0.009 ± 0.001 |
| mIIaQQQ+ EDTA | I-2581 | 1.54 ± 0.03 | 1.64 ± 0.03 | 0.52 ± 0.01 | 0.48 ± 0.01 | 18.8 ± 0.1 | 0.24 ± 0.03 | 12.8 ± 1.6 | 1.68 ± 0.13 | 1.64 ± 0.11 | 976 ± 71 | 0.014 ± 0.001 |
| mIIaQQQ-Δ43 | I-2581 | 2.35 ± 0.07 | 2.15 ± 0.06 | 0.48 ± 0.01 | 0.52 ± 0.02 | 25.8 ± 0.4 | 0.31 ± 0.01 | 12.0 ± 0.3 | 1.67 ± 0.08 | 1.19 ± 0.3 | 713 ± 181 | 0.015 ± 0.004 |
a Fraction of enzyme in the e and E forms in the initial equilibrium are denoted by fe and fE.
b KE,P = k-2/k+2 is the equilibrium dissociation constant for P binding to E. Ke,P = k-3/k+3 is the equilibrium dissociation constant for P binding to e and KConf = [eP]/[EP]. Propagation of errors in the rate constants was used to estimate confidence limits in the calculated equilibrium constants (30).
c NA, not applicable.