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  • Title: Assembly of the prothrombinase complex enhances the inhibition of bovine factor Xa by tick anticoagulant peptide.
    Author: Krishnaswamy S, Vlasuk GP, Bergum PW.
    Journal: Biochemistry; 1994 Jun 28; 33(25):7897-907. PubMed ID: 8011652.
    Abstract:
    The interaction of factor Xa with factor Va on a membrane surface results in the assembly of the prothrombinase complex. The highly specific and multistep interaction between recombinant tick anticoagulant peptide (rTAP) and factor Xa was used to probe perturbations in the macromolecular interaction sites of factor Xa that accompany prothrombinase assembly. Steady-state kinetic studies indicated that the incorporation of factor Xa into prothrombinase resulted in a modest 3-fold increase in the rate constant for inhibition by rTAP. However, the overall dissociation constant for the enzyme-inhibitor interaction (Ki*) was decreased approximately 30-fold to 5.3 pM. This finding was verified by fluorescence stopped-flow studies of the multistep reaction between rTAP and solution-phase factor Xa or prothrombinase by using 4-aminobenzamidine. The second-order rate constant for the binding or rTAP to the protease (k + 1 = 3.35 x 10(6) M-1.s-1) was increased approximately 2-fold (k + 1 = 6.6 x 10(6) M-1.s-1) following the assembly of prothrombinase, while the rate constant for the subsequent slow displacement of the fluorophore from the active site of factor Xa was decreased by 20-fold. Therefore, factor Va alters macromolecular interaction sites on factor Xa; which leads to the stabilization of intermediates in the reaction of the protease with rTAP and an increased overall affinity for the inhibition of factor Xa. Fluorescence measurements of prothrombinase assembly using factor Xa modified with dansylglutamylglycinylarginine chloromethyl ketone (DEGR-Xa) indicated that the preformed rTAP-Xa binary complex bound to factor Va more tightly (Kd = 30.7 +/- 6.2 pM) than factor Xa alone (Kd = 1.25 +/- 0.29 nM). The 30-fold higher affinity of the rTAP-Xa complex for factor Va can completely account for the increased affinity of rTAP for prothrombinase and implies adequate thermodynamic description of the reactions involved. Collectively, the data suggest that the interaction of factor Xa with factor Va on a membrane surface alters macromolecular recognition sites on factor Xa involved in binding rTAP. As a result of this conformational change, the inhibition of factor Xa by rTAP is thermodynamically favored when the enzyme is assembled in the prothrombinase complex.
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