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J Biol Chem, Vol. 274, Issue 35, 24953-24958, August 27, 1999

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

Thung-S. Lai, Thomas F. Slaughter^§, Keith A. Peoples^§, and Charles S. Greenberg^¶

From the Departments of  Medicine, ^¶ Pathology, and ^§ Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710

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 K_act values 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 K_act 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.

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