Thermodynamic Linkage between the S1 Site, the Na+Site, and the Ca2+ Site in the Protease Domain of Human Coagulation Factor Xa

Abstract

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...

The Critical Role of the 185–189-Loop in the Factor Xa Interaction with Na+ and Factor Va in the Prothrombinase Complex

“…These results suggest that similar to thrombin, the conformations of the 186-loop and the 225-loop are allosterically coupled, and thus the dramatic impairment in the amidolytic activity of the K186A mutant may also be caused by its inability to effectively interact with Na ϩ . These results are also in an agreement with previous thermodynamic linkage analysis that demonstrated that the binding of Na ϩ to the 225-loop of fXa allosterically modulates the specificity of the S1 site (Asp 189 ) interaction with the P1-Arg of substrates (16,17). To determine whether the mutagenesis of the 186-loop influences the S1 site specificity of the mutants, their ability to bind to the S1 site specific probe of the trypsin-like serine proteases PAB was examined.…”

“…For instance, the binding of both Ca 2ϩ to the 70-loop and Na ϩ to the 225-loop is known to markedly promote the activity of fXa toward synthetic substrates and natural target inhibitors (14,15). Recent thermodynamic linkage analyses have indicated that both of these metal ion binding sites are energetically linked in fXa (15)(16)(17). Moreover, the same studies have demonstrated that the Na ϩ and the S1 sites of fXa are also allosterically linked (16,17).…”

“…The K d(app) values were calculated from the hyperbolic increase in the rate of substrate hydrolysis as a function of increasing concentrations of Na ϩ as described (15). It has been previously demonstrated that the amidolytic activity of fXa is not dependent on the ionic strength of the reaction buffer (15,17). Thus, no compensating chloride salt was added to the reactions.…”

“…Recent thermodynamic linkage analyses have indicated that both of these metal ion binding sites are energetically linked in fXa (15)(16)(17). Moreover, the same studies have demonstrated that the Na ϩ and the S1 sites of fXa are also allosterically linked (16,17). The S1 site of fXa and other coagulation proteases is a conserved Asp residue at position 189 that is known to interact with a basic residue of all substrates and inhibitors and thus is responsible for determining the P1-Arg specificity of these proteases (11,18).…”

Is it possible to transform trypsin to thrombin?

“…Na þ binds at a different site below the primary specificity pocket (S1) of the enzyme and is coordinated by residues neither directly involved in catalysis nor substrate recognition. In the presence of Na þ , activity of coagulation factors VIIa (10), IXa (11) and Xa (12) and activated protein C (13) is enhanced 3-to 10-fold. The effect of Na þ on thrombin is more pronounced (30-fold increase in activity) (14) and also influences substrate selectivity.…”