The Affinity of Factor X for the Intrinsic Xase Is Determined By Exosite Interactions Between the Substrate and the Enzyme Complex

Distinctive protein substrate specificities are achieved by the membrane-bound enzyme complexes of coagulation, despite the comparable active site geometries of the structurally homologous, trypsin-like coagulation proteinases. In contrast to long-standing ideas in the field, there is increasing evidence for the prevalence of a binding strategy in which binding specificity for the protein substrate is achieved by exosite interactions between sites on the substrate removed from the cleavage site and sites on the enzyme complex distant from the active site of the proteinase. This is evident as a marked disconnect between the kinetic signatures of reversible inhibition of proteinase active site function versus protein substrate cleavage. Despite the widespread clinical interest in factor X (FX) activation by intrinsic Xase composed of the activated antihemophilic factors VIIIa and IXa (FIXa) assembled on membranes, the molecular basis for substrate specificity of the enzyme complex remains poorly understood. This partly reflects the difficulties in assessing active site function of FIXa within intrinsic Xase in a kinetically informative way and in the absence of added alcohols. Here, we exploit the observation that the fluorogenic peptidyl substrate H-D-Leu-Phenylglycine-Arg-aminomethylcoumarin (PF-3688) exhibits an experimentally accessible K_m for FIXa or intrinsic Xase (220 µM and 560 µM, respectively) in the absence of alcohols. We have probed the contribution of active site interactions to intrinsic Xase function using the reversible inhibitor 4-aminobenzamidine (PAB) along with recombinant variants of FX. As expected but never previously shown, PAB acted as a classical competitive inhibitor (K_i ~ 100 µM) of PF-3688 cleavage by intrinsic Xase. The signature features of classical competitive inhibition are that increasing concentrations of inhibitor systematically increase K_m without affecting V_max and that inhibition can be completely overcome at saturating substrate concentrations. This implies that PAB and PF-3688 bind in a mutually exclusive way to the active site of FIXa within intrinsic Xase and is fully expected given the established ability of PAB to reversibly occlude the primary specificity pocket of trypsin-like serine proteinases and the limited way that PF3688 is expected to engage the active site of FIXa. On the other hand, PAB was found to act as a noncompetitive inhibitor for FX activation with increasing concentrations of inhibitor decreasing the V_max for FXa formation and minimally affecting the K_m for FX. In contrast to the peptidyl substrate, this indicates that binding interactions between FX and the active site of FIXa within intrinsic Xase contributes in a minor way to protein substrate affinity. Because FX must dock at the active site for it to be cleaved, the data suggest a multistep binding pathway in which the initial interaction between FX and intrinsic Xase occurs at exosites distant from the active site followed by active site docking and bond cleavage. The initial binding step dominates substrate affinity and the second docking step contributes to the Vmax. These ideas were tested using FX_S195A as an alternate protein substrate in which the catalytic triad Serine is replaced with Alanine to yield a catalytically inactive product, and using FX_R15Q in which the P₁ Arginine is replaced with Glutamine to yield an uncleavable substrate that cannot engage the active site. Both variants were found to act as classical competitive inhibitors of FX activation with K_i ~ K_m. Thus, competitive inhibition of FX activation requires the occlusion of exosite binding by FX and can be accomplished even by a FX variant that cannot engage the active site of FIXa within intrinsic Xase. These findings establish exosite binding as a primary determinant of the affinity and binding specificity of FX for intrinsic Xase. Whether this is entirely accomplished by protein-protein contacts between the substrate and the enzyme complex or also involves contributions from FX binding to membranes remains to be established. Our findings provide new mechanistic insights into intrinsic Xase function that might be exploited to modulate FXa formation through the intrinsic pathway of coagulation in human disease.