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 Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) 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, 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) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule 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.
Lai
T S
TS
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Slaughter
T F
TF
Peoples
K A
KA
Greenberg
C S
CS
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013917:Thrombin / Q000378:metabolism; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics*
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D011503:Transglutaminases; D013917:Thrombin; D002118:Calcium