Factor VIIa residue Arg290 is required for efficient activation of the macromolecular substrate factor X.
The serine protease factor VIIa (VIIa) in complex with tissue factor is responsible for initiating proteolytic events in the coagulation pathways. Efficient proteolysis by the extrinsic activation complex appears to depend on structural determinants in the cofactor as well as the light and heavy chain domains of VIIa. This study characterizes the functional defect resulting from alanine replacement for R290 in the VIIa protease domain. VIIa R290-->A bound both full-length and soluble tissue factor with affinities indistinguishable from wild-type VIIa, consistent with overall unaltered folding of the mutant protein. The catalytic function of VIIa R290-->A was further demonstrated to be unperturbed when analyzed with three different peptidyl p-nitroanilide substrates, indicating that the function of the catalytic triad is not affected by the mutation. However, proteolytic activation of factor X was diminished due to a 4-5-fold decreased kcat in the presence and a > 10-fold decreased rate in the absence of a negatively charged phospholipid surface. The functional defect resulting from the R290-->A replacement was observed in the presence and absence of cofactor. Within the structural framework of serine protease domains, R290 is predicted to be localized in a surface-exposed loop suggested to contribute to substrate selectivity in other serine proteases, consistent with the proposed functional role of R290 in the proteolytic activation of the natural substrate factor X.
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Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
D000577:Amides / Q000378:metabolism; D001120:Arginine / Q000235:genetics; D015942:Factor VIIa / Q000235:genetics / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000096:biosynthesis*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013925:Thromboplastin / Q000378:metabolism
D000577:Amides; D011506:Proteins; D011994:Recombinant Proteins; D005170:Factor X; D013925:Thromboplastin; D001120:Arginine; D015942:Factor VIIa; D015951:Factor Xa