The importance of specific gamma-carboxyglutamic acid residues in prothrombin. Evaluation by site-specific mutagenesis.
Prothrombin has 10 gamma-carboxyglutamic acid residues which are essential for the metal ion binding properties and membrane binding function of the protein. To assess the importance of each gamma-carboxyglutamic acid residue we made, by site directed mutagenesis, a series of mutant human prothrombins each with a single glutamic acid to aspartic acid substitution at positions 6, 7, 14, 16, 19, 20, 25, 26, 29, or 32 which are gamma-carboxylated in native prothrombin. Along with wild-type prothrombin, the prothrombin mutants were expressed in Chinese hamster ovary cells, purified by immunoaffinity chromatography using polyclonal anti-prothrombin antibodies, and shown by amino acid analysis to contain the expected number of gamma-carboxyglutamic acid residues. Only substitution of gamma-carboxyglutamic acid 6 with aspartic acid yields a protein with procoagulant activity, affinity for phospholipid and KM(app) for prothrombinase indistinguishable from wild-type prothrombin. In contrast, the conservative gamma-carboxyglutamic acid to aspartic acid mutation at positions 16, 26, or 29 results in proteins with little or no procoagulant activity, Kd(app) for binding to phospholipid at least 200-fold higher than wild-type prothrombin and a KM(app) for interaction with the prothrombinase complex nearly 100-fold higher than wild-type prothrombin. The mutations at residues 7, 14, 19, 20, 25, or 32 yielded proteins with intermediate procoagulant activities, between 15 and 52% of wild-type prothrombin. These data have been interpreted to suggest that there are certain gamma-carboxyglutamic acid residues which are important to maintaining the basic structure of the calcium-liganded Gla domain while other gamma-carboxyglutamic acid residues subserve other functions including membrane binding and interdomain interactions.
Ratcliffe
J V
JV
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
Furie
B
B
Furie
B C
BC
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011516:Prothrombin / Q000235:genetics / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism
D010743:Phospholipids; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D013925:Thromboplastin