IDENTIFICATION OF SURFACE RESIDUES MEDIATING TISSUE FACTOR-BINDING AND CATALYTIC FUNCTION OF THE SERINE-PROTEASE FACTOR VIIA

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a compr ehensive analysis of the functional surface of Wa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which co ntribute to TF binding and factor X hydrolysis. Energetically importan t binding contacts at the interface with TF were identified in the fir st epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg -79) and in the protease domain (Arg-277, Met-306, Asp-309). The obser ved energetic defects are in good agreement with the corresponding res idues in TF, suggesting that the VIIa light chain plays a prominent ro le in high affinity binding of cofactor. Mutation of protease domain i nterface residues indicated that TF allosterically influences the acti ve site of VIIa. Stabilization of a labile zymogen to enzyme transitio n could explain the activating effect of TF on Wa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, a nd (iii) a large electronegative exosite which is in a position analog ous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF-VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF-VIIa with macromolecular s ubstrate.