In Vitro Mutagenesis Study of Two Critical Glutamic Acids in the Calcium Binding Loop of the Factor IX Heavy Chain

Summary

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 IX_Glu235Lys and factor IX_Glu245Lys appeared normal by polyacrylamide gel analysis. (Factor IX_Glu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IX_Glu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IX_{Glu235&245Lys}: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IX_Glu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IX_Glu245Lys and factor IX_{Glu235&245Lys} failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IX_Glu235Lys and factor IX_Glu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium’s absence, factor IX_Glu235Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IX_Glu245LyS’s conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium. We conclude that glutamic acids 235 and 245 are, as predicted, involved in the calcium binding site in the factor IX catalytic domain, and that the correct conformation of this calcium binding loop is essential for maintaining the active site pocket.

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