Related Experiment Videos
Asp-70-->Lys mutant of factor X lacks high affinity Ca2+ binding site yet retains function
1Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City.
The Journal of Biological Chemistry
|August 26, 1994
Summary
This study investigates calcium binding sites in factor X, revealing their importance in activation complexes. Mutations suggest structural similarities between factor X and trypsin calcium binding sites.
Area of Science:
- Biochemistry
- Molecular Biology
- Hemostasis
Background:
- Factor X is a key serine protease in the coagulation cascade.
- Calcium ions play a critical role in the assembly and function of coagulation factor activation complexes.
- The precise role of specific calcium binding sites within factor X itself remains incompletely understood.
Purpose of the Study:
- To elucidate the function of a putative high-affinity calcium binding site in the protease domain of factor X.
- To investigate the calcium-dependent activation of factor X and its role in prothrombin activation.
- To explore the structural similarities between calcium binding sites in factor X and other proteases like trypsin.
Main Methods:
- Preparation of deletion mutants (E2FX) and point mutants (E2FXD70K, E2FXE80K) of factor X.
- Characterization of calcium binding affinities (Kd) and functional activity of the mutants.
- Assays for the calcium-dependent activation of E2FXD70K and E2FX by tissue factor-factor VIIa and factor Va complexes.
- Analysis of prothrombin and prethrombin 1 activation kinetics.
Main Results:
- Mutants E2FXD70K and E2FXE80K retained functional activity but showed altered calcium binding properties.
- The D70K mutation abolished high-affinity calcium binding and Ca(2+)-dependent substrate activation.
- Activation of E2FXD70K by tissue factor-factor VIIa and factor Va remained Ca(2+)-dependent, but with reduced affinity for calcium.
- Activation of prethrombin 1 by E2FXaD70K was Ca(2+)-independent in the absence of factor Va, unlike wild-type E2FXa.
Conclusions:
- The results indicate the presence of functionally significant calcium binding sites beyond factor X in activation complexes.
- The study suggests structural homology between the calcium binding sites of factor X and trypsin.
- Specific mutations within the factor X calcium binding site may confer calcium-independent function through internal salt bridge formation.