Residues Y179 and H101 of a hydrophobic patch of factor VII are involved in activation by factor Xa

We studied factor Xa activation of human factor VII in hopes of identifying factor VII residues, not adjacent to the cleavage site, involved in this i nteraction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with mult iple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X acti vation, and the residues of MM4 are located at a hydrophobic patch of facto r VII on the opposite side of the catalytic domain from those in MM3. Only H101A, Y179A, and MM4 were activated significantly more slowly than the wil d type. Results of our kinetic analyses showed that the catalytic efficienc y of factor Xa for activation of factor VII was 176- and 234-fold higher th an that for H101A and Y179A, respectively. All the mutants with measurable activity had affinities for tissue factor similar to those of the wild type . The activated hydrophobic patch residues, except N100A, which is adjacent to one of the catalytic residues, had normal activities toward both a smal l peptide substrate and factor X. The rest of the activated mutants (except D102Q with no activity) had reduced activities toward the small substrate (except R147A) and factor X. We conclude that factor VII activation by fact or Xa and factor VIIa's catalytic interaction with factor X involve differe nt regions in the catalytic domain, and residues H101 and Y179, part of an aromatic hydrophobic patch, are specifically involved in factor Xa activati on of factor VII.