The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na ؉ binds to the substrate bound FXa with K d ϳ39 mM in the absence and ϳ9.5 mM in the presence of Ca 2؉ . Sodium-bound FXa (sodium-Xa) has ϳ18-fold increased catalytic efficiency (ϳ4.5-fold decrease in K m and ϳ4-fold increase in k cat ) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca 2؉ further increases this k cat ϳ1.4-fold. Ca 2؉binds to the protease domain of substrate bound FXa with K d ϳ705 M in the absence and ϳ175 M in the presence of Na ؉ . Ca 2؉ binding to the protease domain of FXa (Xa-calcium) has no effect on the K m but increases the k cat ϳ4-fold in hydrolyzing S-2222, and Na ؉ further increases this k cat ϳ1.4-fold. In agreement with the K m data, sodium-Xa has ϳ5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and ϳ4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca 2؉ (؎Na ؉ ) has no effect on these interactions. Antithrombin binds to Xa-calcium with a ϳ4-fold faster rate, to sodium-Xa with a ϳ24-fold faster rate and to sodium-Xa-calcium with a ϳ28-fold faster rate. Thus, Ca 2؉ and Na ؉ together increase the catalytic efficiency of FXa ϳ28-fold. Na ؉ enhances Ca 2؉ binding, and Ca 2؉ enhances Na ؉ binding. Further, Na ؉ enhances S1 site occupancy, and S1 site occupancy enhances Na ؉ binding. Therefore, Na ؉ site is thermodynamically linked to the S1 site as well as to the protease domain Ca 2؉ site, whereas Ca 2؉ site is only linked to the Na ؉ site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.Factor X is a vitamin K-dependent plasma glycoprotein that plays a crucial role in blood coagulation. The human protein circulates as a zymogen with a molecular weight of ϳ59,000 and consists of a light chain (amino acids 1-139) and a heavy chain (amino acids 143-448) held together by a single disulfide bond between Cys-132 and Cys-302 (1). Upon activation by factor VIIa, Ca 2ϩ , and tissue factor or by factor IXa, Ca 2ϩ phospholipid, and factor VIIIa, a single peptide bond in factor X between residues Arg-194(c15) 1 and Ile-195(c16) is cleaved with resultant formation of a serine protease, factor Xa (FXa), 2 and release of a 52-residue activation peptide (1, 2). FXa is converted to its -form where a ϳ4-kDa peptide is cleaved off from the COOH terminus of the heavy chain; this, however, does not result in a loss of coagulant activity (3). FXa converts prothrombin to thrombin (IIa) in a reaction requiring Ca 2ϩ , phospholipid membrane, and factor Va (FVa) (2, 4). Through its active site Ser-397(c195), FXa also binds to the serpin antithrombin (AT) (5) and to the second Kunitz domain of tissue factor pathway inhibitor (TFPI) (6).The NH 2 termi...
Human factor X is a two-chain, 58-kDa, vitamin K-dependent blood coagulation zymogen. The light chain of factor X consists of an NH 2 -terminal ␥-carboxyglutamic acid (Gla) domain, followed by a few helical hydrophobic residues and the two epidermal growth factor-like domains, whereas the heavy chain contains the serine protease domain. In this study, native factor X was found to contain three classes of Ca Factor X is a vitamin K-dependent multidomain protein that participates in the middle phase of blood coagulation (1). Factor X is essential for hemostasis since a reduction in its functional activity results in a rare autosomal recessive bleeding disorder known as Stuart-Prower factor deficiency (2). The human protein is synthesized in the liver as a precursor molecule of 488 amino acids (3). The amino-terminal 40 amino acids constitute the prepro leader sequence, which is removed prior to secretion of the molecule. Additionally, during biosynthesis, the protein undergoes several posttranslational modifications including glycosylation, ␥-carboxylation (of the first 11 glutamic acid residues), -hydroxylation (of Asp-63), and removal of a tripeptide (Arg-Lys-Arg) between Arg-139 and Ser-143 (3). The resulting mature protein is a zymogen of serine protease factor Xa and consists of a light chain (amino acids 1-139) and a heavy chain (amino acids 143-448) held together by a single disulfide bond between Cys-132 and Cys-302.Gene arrangement, amino acid sequence, and modular structure of factor X strongly suggest that the protein is organized into several distinct domains (3, 4). The amino terminus of the light chain contains 11 ␥-carboxyglutamic acid (Gla) 1 residues and represents the Gla domain (residues 1-39) of factor X (3). The Gla domain is followed by a short hydrophobic stack (residues 40 -45) and two epidermal growth factor (EGF)-like domains (residues 46 -84 (EGF1) and residues 85-128 (EGF2)). The amino terminus of the heavy chain of factor X contains the activation peptide region of 52 amino acids (residues 143-194) followed by the serine protease domain of 254 amino acids (residues 195-448), which features the active site triad of His-236(57), 2 Asp-282(102), and Ser-379(195) (3).* This work was supported in part by National Institutes of Health Grants HL36365 (to S. P. B.) and HL25942 (to A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported in part by a senior postdoctoral fellowship from the American Heart Association, Missouri Affiliate.* 1 The abbreviations and trivial names used are: Gla, ␥-carboxyglutamic acid; DEGR-CK, dansyl-Glu-Gly-Arg-chloromethyl ketone; EGF, epidermal growth factor; des-44-X or -Xa, Gla-domainless human factor X or Xa; PC, phosphatidylcholine; PS, phosphatidylserine; BSA, bovine serum albumin; PL, phospholipid; p-AB, p-aminobenzamidine; peptide 1, peptide containing the sequence of...
Previous studies revealed that cleavage at Arg-318 -Ser-319 in the protease domain autolysis loop of factor IXa results in its diminished binding to factor VIIIa. Now, we have investigated the importance of adjacent surface-exposed helix 330 -338 (162-170 in chymotrypsin numbering) of IXa in its interaction with VIIIa. IX WT , eight point mutants mostly based on hemophilia B patients, and a replacement mutant (IX helixVII in which helix 330 -338 is replaced by that of factor VII) were expressed, purified, and characterized. Each mutant was activated normally by VIIa-tissue factor-Ca 2؉ or XIa-Ca 2؉ . However, in both the presence and absence of phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IXa EGF1 domain in binding to VIIIa was also examined. Two mutants (IX Q50P and IX PCEGF1 , in which EGF1 domain is replaced by that of protein C) were used. Strikingly, interactions of the activated EGF1 mutants with VIIIa were impaired only in the presence of phospholipid. We conclude that helix 330 in IXa provides a critical binding site for VIIIa and that the EGF1 domain in this context primarily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with VIIIa.
binding to the protease domain increases the S1 reactivity ϳ3-fold and prevents proteolysis in the autolysis loop; and (d) proteolysis in the autolysis loop leads to a loss of catalytic efficiency with retention of S1 binding site and a further ϳ8-fold reduction in affinity of factor IXa for factor VIIIa.
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