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

Abstract

The S1 site (Asp 189 ) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp 185 , Lys 186 , and Glu 188 ) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly (ϳ2-fold) and dramatically (ϳ20 -50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na ؉ has also been altered, with a modest impairment (ϳ2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an ϳ6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na ؉ loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na ؉ -binding site of the protease.Factor X is a vitamin-K dependent serine protease zymogen in plasma that upon activation to factor Xa (fXa) 1 forms a high affinity complex with other components of the prothrombinase complex (factor Va, negatively charged phospholipid vesicles, and calcium) to activate prothrombin to thrombin during the blood coagulation process (1-5). The catalytic efficiency of fXa in the prothrombinase complex is five orders of magnitude higher than that of fXa alone (1, 2). Most of the accelerating effect of the prothrombinase complex assembly is attributed to the cofactor function of factor Va (fVa), which can bind to and alter the kinetic properties of fXa by inducing conformational changes in the structure of the protease and also by providing secondary binding sites for the substrate prothrombin in the activation complex (6 -10). The molecular details of the fXa interaction with fVa have not been fully elucidated. However, recent results have indicated that the mutagenesis of the basic residues of the heparin binding 162-helix on the protease domain (especially Arg 165 in the chymotrypsinogen numbering system (11)) dramatically impairs the ability of fXa to interact with fVa in the prothrombinase complex (12). In addition to the 162-helix, other basic residues of the heparin binding site of fXa have been implicated in fVa binding (12, 13); however, the exact contribution of these residues to the recognition mechanism of the protease-cofactor interaction has not been studied in detail.Similar to other coagulation proteases, fXa is an allosteric enzyme, and thus the binding of metal ions an...

Role of the α-Helix 163-170 in Factor Xa Catalytic Activity

“…1). Furthermore there is some evidence that both the FVa-and Na ϩ -binding sites of FXa are energetically linked (20,23). Altogether these observations suggest that there is an allosteric linkage between the Na ϩ -binding site and FVa-binding h163-170.…”

“…It has been demonstrated previously that the amidolytic activity of FXa is not dependent on the ionic strength of the reaction buffer in the range of NaCl used (19,23,30). Thus, no compensating chloride salt was added to the reactions.…”

Mutant N143P Reveals How Na+ Activates Thrombin

“…Curiously, the original observation on thrombin was at odds with the accepted view at the time that salts, and Na ϩ in particular, actually acted as inhibitors of thrombin hydrolysis of synthetic substrates (17,18). In fact, the inhibitory effect of Na ϩ was rationalized as a direct perturbation of the catalytic His 57 that would reduce effectiveness of the charge relay system (18). Wells and Di Cera demonstrated that the Na ϩ activation of thrombin is specific and allosteric and involves the transition of the enzyme between two active forms (19) leading to an increase in k cat and a decrease in K m .…”

The Conformational Switch from the Factor X Zymogen to Protease State Mediates Exosite Expression and Prothrombinase Assembly

“…More recent studies have shown that parts of this helical region appear linked to the S1-and Na ϩ binding sites (56), lending further support to the idea that changes in the activation domain will likely impact FVa binding. Furthermore, mutagenesis studies have shown that the Na ϩ binding site and the 185-189 loop are important for FVa binding (19,23,24,57 . Aliquots of the reaction mixture were quenched during the initial rate of the reaction (0, 0.5, 1, 1.5, and 2.0 min), and thrombin generation was determined by using the chromogenic substrate S-2238.…”