Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 5

Serine Protease Domain

The C-terminal portion of factor IX demonstrates marked sequence homology with zymogens of serine proteases, such as trypsin and chymotrypsin. This region is almost 250 amino acids long and contains, in latent form, the enzyme active site of factor IXa. The mechanism of enzyme activation involves a process known as limited proteolysis. On cleavage of peptide bonds, the proenzyme is rapidly converted to its enzyme form. The cleavage of two peptide bonds in factor IX leads to the generation of the enzyme factor IXa. The enzyme active site is common to all serine protease and contains the enzymatic machinery for the hydrolysis of peptide bonds. Superimposed on this generic structure is an extended substrate binding site, surrounding the active site, that defines the high substrate specificity of factor IXa toward protein substrates.

Calcium-Binding Properties

Factor IX binds to metal ions. As with the other vitamin K-dependent proteins, there appear to be two classes of metal-binding sites: high affinity and lower affinity. MLID79005660  82 The g-car- boxyglutamic acid domain defines some, but not all, of these metal-binding sites, since factor IX chemically modified by the removal of the g-carboxyglutamic acid domain still retains a high-affinity calcium-binding site. MLID84185715  68 Similar findings have been demonstrated in factor IXa. MLID85279476  83 A calcium-binding site has been localized to the first EGF domain of factor IX. MLID90151623  84 Asp 47, Asp 49, Gln 50, and Asp 64 are involved in calcium binding within the first EGF domain. MLID91232585  85 A second low-affinity metal binding site may be present in the region of Phe 77. MLID90151623 MLID91232585  84,85 Astermark et al. MLID91115865  86 have demonstrated that while the g-carboxyglutamic acid and EGF domains can bind calcium ions independently, the intact g-carboxyglutamic acid-EGF domains bind calcium ions with higher affinity. MLID91115865  86 Interdomain contact and stabilization were also detected in that thermal denaturation of the g-carboxyglutamic acid-EGF modules is 10єC higher then the g-carboxyglutamic acid domain alone. MLID93231988  87 A high-affinity calcium-binding site has also been described in the heavy chain of factor IX involving residues 235, 237, 240, and 245. MLID92108008  88

Two classes of metal-binding sites have been identified in both factor IX and prothrombin using conformation-specific antibodies. MLID87222379 MLID87033727  89,90 Occupation of these two classes of metal-binding sites leads to two sequential conformational changes in these proteins. The first conformational change, induced by magnesium and many other divalent cations, is associated with the quenching of intrinsic fluorescence. The second, metal-selective conformational change is only induced by calcium ions. Fab fragments of the conformation-specific antibodies, directed against factor IX after it has undergone the conformational transition that is calcium ion selective, block the binding of factor IX to phospholipid vesicles and the activation of factor IX by factor XIa. This finding suggests that the conformer achieved only in the presence of calcium is necessary for the expression of a phospholipid-binding site and for the binding of factor IX by factor XIa.

Membrane-Binding Properties

The interaction of factor IX and factor IXa with phospholipid membranes has been studied using phosphatidylserine/phosphatidylcholine vesicles. MLID86131633  76 Factor IX binds to vesicles in the presence of Ca2+, but not in the presence of Mg2+ or EDTA. Furthermore, des-g-carboxy factor IX, prepared from the plasma of patients who are given warfarin, does not interact with phospholipid vesicles even in the presence of Ca2+. These results emphasize the importance of the g-carboxyglutamic acid-rich region in defining phospholipid-binding properties. The specificity of factor IX for an acidic phospholipid (e.g., phosphatidylserine) may parallel other vitamin K-dependent proteins that have been evaluated using phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, or the specificity may be different. Factor IX affinity for phosphatidyl- choline-phosphatidylserine membranes is independent of the phosphatidylserine concentration with phosphatidylserine compositions >20–30% MLID87075663  91; a Kd of about 1–2 mM has been measured. The expression of phospholipid-binding properties involves two metal-dependent conformational transitions. MLID87222379  89 Like factor IX, factor IXa binds to phospholipid vesicles in the presence of calcium ions. Experiments in which Trp 42 is mutated suggest that the aromatic amino acid stack may also play a role in the binding of factor IX to phospholipid vesicles. MLID93185650  92,93