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 interaction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with multiple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X activation, and the residues of MM4 are located at a hydrophobic patch of factor 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 wild type. Results of our kinetic analyses showed that the catalytic efficiency of factor Xa for activation of factor VII was 176- and 234-fold higher than that for H101A andY179A, 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 small 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 factor Xa and factor VIIa's catalytic interaction with factor X involve different regions in the catalytic domain, and residues H101 and Y179, part of an aromatic hydrophobic patch, are specifically involved in factor Xa activation of factor VII.
Jin
J
J
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Chang
J
J
Stafford
D W
DW
Straight
D L
DL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D003196:Computer Graphics; D017124:Conserved Sequence; D005167:Factor VII / Q000737:chemistry* / Q000378:metabolism*; D015942:Factor VIIa / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
D011994:Recombinant Proteins; D005167:Factor VII; D002918:Chymotrypsin; D015942:Factor VIIa; D014357:Trypsin; D015951:Factor Xa