Back to Search
Start Over
Defining the Factor Xa-binding Site on Factor Va by Site-directed Glycosylation
- Source :
- Journal of Biological Chemistry. 277:50022-50029
- Publication Year :
- 2002
- Publisher :
- Elsevier BV, 2002.
-
Abstract
- Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa.FXa complex assembly.
- Subjects :
- Models, Molecular
DNA, Complementary
Glycosylation
Time Factors
Protein Conformation
Biochemistry
Thromboplastin
chemistry.chemical_compound
Thrombin
Prothrombinase
Consensus sequence
medicine
Animals
Humans
Binding site
Molecular Biology
Phospholipids
Binding Sites
Dose-Response Relationship, Drug
Tunicamycin
Mutagenesis
Cell Biology
Recombinant Proteins
Anti-Bacterial Agents
Protein Structure, Tertiary
Kinetics
chemistry
Factor Va
COS Cells
Factor Xa
Mutagenesis, Site-Directed
Electrophoresis, Polyacrylamide Gel
Protein Binding
medicine.drug
Tenase
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 277
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....b8b952f0152f6031bff5b188fddf6754
- Full Text :
- https://doi.org/10.1074/jbc.m205609200