Tissue Factor residue Asp44 regulates catalytic function of the bound proteinase Factor VIIa.
The coagulation pathways are initiated by the cell-surface receptor Tissue Factor (TF), which binds the serine proteinase coagulation Factor VIIa (VIIa), resulting in enhanced catalytic function, both amidolytic, towards small pseudo-substrates, and proteolytic, towards macromolecular substrates. Here we implicate Asp44 in TF as a ligand-interactive residue that, in contrast with previously characterized binding residues, is involved in the enhancement of VIIa catalytic function. Whereas charge neutralization by replacement of Asp44 with Asn did not reduce function of human TF, the exchange by Ala resulted in mutants with 8-fold reduced affinity for binding of VIIa. Enhancement of VIIa amidolytic function by TF Ala44 was reduced by 20-25% relative to wild-type and support of proteolytic function was diminished 6-fold indicating that this cofactor residue is significantly enhancing proteolysis of the macromolecular substrate by VIIa. Replacement of Asp44 by Glu, Thr, and Arg exhibited a less severe phenotype with an approx. 4-fold reduced affinity for VIIa and a 2-3 fold diminished activation of Factor X. The improved activity of these mutants as compared with the Ala replacement is consistent with functional importance of an extended side chain at this position. The specific influence of the Asp44 exchange on catalytic function of the TF x VIIa complex indicates fine specificity of the TF ligand interface in mediating receptor and cofactor function.
Kelly
C R
CR
Department of Immunology and Vascular Biology, The Scripps Institute IMM-17, La Jolla, CA 92937, U.S.A.
Schullek
J R
JR
Ruf
W
W
Edgington
T S
TS
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001667:Binding, Competitive; D016466:CHO Cells / Q000378:metabolism / Q000502:physiology; D002384:Catalysis; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D015942:Factor VIIa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D001224:Aspartic Acid; D004247:DNA; D013925:Thromboplastin; D010447:Peptide Hydrolases; D015942:Factor VIIa; D000409:Alanine