Abstract
Factor X activation by the intrinsic Xase complex, composed of factor IXa bound to factor VIIIa on membranes, is essential for the amplified blood coagulation response. The biological significance of this step is evident from bleeding arising from deficiencies in factors VIIIa or IXa in hemophilia. Here, we assess the mechanism(s) that enforce the distinctive specificity of intrinsic Xase for its biological substrate. Active-site function of IXa was assessed with a tripeptidyl substrate (PF-3688). The reversible S1 site binder, 4-aminobenzamidine (pAB), acted as a classical competitive inhibitor of PF-3688 cleavage by Xase. In contrast, pAB acted as a noncompetitive inhibitor of factor X activation. This disconnect between peptidyl substrate and protein substrate cleavage indicates a major role for interactions between factor X and extended sites on Xase in determining substrate affinity. Accordingly, an uncleavable factor X variant, not predicted to engage the active site of IXa within Xase, acted as a classical competitive inhibitor of factor X activation. Fluorescence studies confirmed the binding of factor X to Xase assembled with IXa with a covalently blocked active site. Our findings suggest that the recognition of factor X by the intrinsic Xase complex occurs through a multistep "dock-and-lock" pathway in which the initial interaction between factor X and intrinsic Xase occurs at exosites distant from the active site, followed by active-site docking and bond cleavage.
Highlights
The serine proteinases of blood coagulation contain catalytic domains that are structurally similar and resemble trypsin, an ancestral member of the S1 peptidase family of which chymotrypsin is the archetype [1,2,3]
As has been reported for the analogous prothrombinase complex, incorporation of IXa into the intrinsic Xase complex yielded a modest decrease in the rate and catalytic efficiency of peptidyl substrate cleavage
Given that the rate of cleavage of the cognate protein substrate is vastly increased following the assembly of prothrombinase or intrinsic Xase, the findings illustrate, once again, an obvious disconnect between the constraints affecting the kinetic constants for protein substrate cleavage relative to active-site function
Summary
Factor IXa exhibits very weak amidolytic activity toward synthetic peptidyl substrates. Initial velocity studies of PF-3688 cleavage by IXa alone (Fig. 1A) or in complex with VIIIa and PCPS (Fig. 1B) in the presence of different fixed concentrations of pAB yielded results consistent with classical competitive inhibition. The kinetic signature of classical competitive inhibition is that increasing concentrations of inhibitor increase Km without affecting Vmax because inhibition at any one inhibitor concentration can be completely overcome by saturating with substrate This result implies that pAB and PF-3688 bind in a mutually exclusive way to the active site of IXa in solution or assembled into intrinsic Xase. Inhibition by pAB arises from a decreased Vmax because some fraction of the enzyme is tied up as E·S·I in the presence of inhibitor regardless of substrate concentration This redistribution decreases the fraction of the E·S* species and thereby the rate of product formation even at saturating concentrations of substrate. This simple kinetic approach suggests a multistep dock-and-lock pathway for kcatobs
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