The influence of factor Va on the active site of factor Xa

Abstract

The interaction of factor Xa with factor Va on the membrane surface results in a 3,000-fold increase in the kcat for the activation of prothrombin catalyzed by factor Xa. The reaction between the transition state irreversible inhibitor dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) and factor Xa was characterized and employed to evaluate changes in the active site of factor Xa resulting from its interaction with factor Va, which may account for the increased catalytic efficiency of the enzyme complex. Inhibition studies indicated a two-step inhibition reaction involving a reversible binding step (Ki = 1.13 microM) and an irreversible alkylation step (ki = 0.65 s-1). The interaction between factors Va and Xa in solution or on membranes resulted in a small decrease in the overall second-order rate constant (ki/Ki) for the inhibition reaction. The incorporation of DEGRck into the active site of factor Xa results in a large change in the fluorescence intensity of the dansyl moiety. The fluorescence change was employed to study the reaction between enzyme and inhibitor directly by stopped-flow fluorescence measurements. The fluorescence traces were biphasic, indicating that the association of DEGRck with factor Xa and the subsequent covalent modification of the active site could be resolved because of differences in fluorescence intensities of the intermediate and product. This interpretation was verified by rapid chemical quench experiments. The reaction between DEGRck and factor Xa was characterized by a second-order association rate constant of 8.38 +/- 0.28 x 10(5) M-1.s-1 and an intrinsic rate constant for the alkylation step of 1.67 +/- 0.25 s-1. The rate constant for the alkylation step was unchanged in the presence of factor Va and membranes, whereas the association rate constant was modestly decreased by approximately 50%. The decrease in the association rate constant did not result from the partitioning of factor Xa to the membrane surface and could therefore be ascribed to an effect of factor Va on the protease. The data suggest that the interaction between factors Va and Xa on the membrane surface does not detectably alter the catalytic residues but may result in changes in the binding or accessibility of substrate to extended macromolecular recognition sites on the protease.