Site-directed mutagenesis of the calcium-binding site of blood coagulationfactor XIIIa

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalentl y ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438) Ala(457) , Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) a nd Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp( 479)) that are in close proximity. Alanine substitution mutants of these re sidues 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 proteo lysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490 A, 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, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490), Ca2+-binding ligands that regulate catalysis, The binding of calcium ion to this site protects the mo lecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.