Site-directed Mutagenesis of the Calcium-binding Site of Blood Coagulation Factor XIIIa

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp438, Ala457, Glu485, and Glu490. We mutated two glutamic acid residues (Glu485 and Glu490...

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Veröffentlicht in:The Journal of biological chemistry 1999-08, Vol.274 (35), p.24953-24958
Hauptverfasser: Lai, Thung-S., Slaughter, Thomas F., Peoples, Keith A., Greenberg, Charles S.
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Sprache:eng
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Zusammenfassung:Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp438, Ala457, Glu485, and Glu490. We mutated two glutamic acid residues (Glu485 and Glu490) and three aspartic acid residues (Asp472, Asp476, and Asp479) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The Kactvalues for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the Kact values were not changed by mutagenesis. However, Asp476 and Asp479 are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu485 and Glu490 are Ca2+-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp476 and Asp479 play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.274.35.24953