ACTIVE-SITE MODIFICATION OF FACTOR VIIA AFFECTS INTERACTIONS OF THE PROTEASE DOMAIN WITH TISSUE FACTOR

In the initiation of coagulation, tissue factor (TF) allosterically ac tivates the serine protease factor VIIa (VIIa) through specific intera ctions with protease domain residues. These interactions, and conseque ntly affinity for TF, may be influenced by conformational changes in t he protease domain that result from zymogen-enzyme transition or occup ancy of the active site by tight binding inhibitors, In functional com petition and direct binding analysis, we determined affinities for zym ogen and enzyme species of wild-type VII and of mutants at protease do main residues that contact TF. We demonstrate that TF binding is not i nfluenced by zymogen activation, indicating that the protease domain o f zymogen and enzyme dock similarly with TF, In contrast, active site occupancy enhanced the affinity for TF by predominantly decreasing the dissociation rate of the TF.VIIa complex. Of the three interface resi dues studied, only Met(306) played a major role in the inhibitor-induc ed increase in affinity. Met(306) is also important for transmitting t he allosteric changes from TF to the active site, resulting in enhance d catalysis. This study thus provides evidence for a bidirectional con formational interdependence of the interface residue Met(306) and the active site of VIIa.