Site-directed mutagenesis but not gamma-carboxylation of Glu-35 in factor VIIa affects the association with tissue factor.
Factor VIIa is a vitamin K-dependent enzyme whose gamma-carboxyglutamic acid (Gla)-containing domain is important for calcium ion-dependent binding to the cofactor tissue factor and membrane surfaces. This domain contains 10 Gla residues, the individual roles and importance of which are not known. Comparisons with the homologous protein C, factor IX and prothrombin may provide functional information on the first nine Gla residues, whereas no data can be extrapolated to Gla-35 in factor VIIa. Therefore, the effects of posttranslational gamma-carboxylation and site-directed mutagenesis of Glu-35 were investigated. Mutations to Asp, Gln or Val all lead to a lower affinity for tissue factor by decreasing the rate of association, in the case of the Val mutant by a factor of 200, as measured by surface plasmon resonance. In contrast, Glu or Gla side chains at position 35 appear to fulfil the functional roles equally well.
Persson
E
E
Vessel Wall Biology, Health Care Discovery, Novo Nordisk AIS, Gentofte, Denmark.
Nielsen
L S
LS
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D017931:DNA Primers; D015942:Factor VIIa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013925:Thromboplastin / Q000378:metabolism*
D017931:DNA Primers; D018698:Glutamic Acid; D015055:1-Carboxyglutamic Acid; D013925:Thromboplastin; D015942:Factor VIIa; D002118:Calcium