title: In vitro mutagenesis study of two critical glutamic acids in the calcium binding loop of the factor IX heavy chain.
abstract: We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium's absence, factor IXGlu245Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245Lys's conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biology, University of North Carolina, Chapel Hill 27599.
authors.forename: N
authors.initials: N
authors.lastname: Hamaguchi
authors.forename: D
authors.initials: D
authors.lastname: Stafford
mesh_terms: D000941:Antigens / Q000097:blood; D001483:Base Sequence; D001665:Binding Sites; D001780:Blood Coagulation Tests; D002135:Calcium-Binding Proteins / Q000235:genetics*; D005164:Factor IX / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D015945:Factor XIa / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics*; D017354:Point Mutation; D013725:Terbium
chemical_list: D000941:Antigens; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D013725:Terbium; D018698:Glutamic Acid; D005164:Factor IX; D015945:Factor XIa