Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking

The upstream coagulation enzymes are homologous trypsin-like serine proteas es that typically function in enzyme-cofactor complexes, exemplified by coa gulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF), TF cooperates with VIIa t o create a bimolecular recognition surface that serves as an exosite for fa ctor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity, Muta tion of the P1 Arg residue in factor X to Gin prevented activation by the T F VIIa complex but did not reduce macromolecular substrate affinity for TF VIIa. Similarly, mutations of the S and S' subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the aff inity for small chromogenic substrates and the efficiency of factor X sciss ile bond cleavage were reduced. Thus, docking of the activation peptide bon d to the catalytic cleft of this enzyme-cofactor complex does not significa ntly contribute to affinity for macromolecular substrate. Rather, it appear s that the creation of an extended macromolecular substrate recognition sur face involving enzyme and cofactor is utilized to generate substrate specif icity between the highly homologous, regulatory proteases of the coagulatio n cascade.