Ordered Cleavage of Prothrombin by Prothrombinase Results from Constrained Binding and Preferential Active Site Engagement by One of the Two Cleavage Sites in Prothrombin.

The conversion of prothrombin to thrombin is a paradigm for proteolytic activation reactions wherein product is produced following cleavage at more than one site in the substrate. Human prothrombinase catalyzes thrombin formation by sequential cleavage of prothrombin at Arg₃₂₀ followed by cleavage at Arg₂₇₁. The largely ordered activation arises because Arg₃₂₀ in intact prothrombin is hydrolyzed by prothrombinase with a Vmax that is ~30-fold greater than that for cleavage at Arg₂₇₁ while substrate affinity is unchanged. Paradoxically, this phenomenon has been proposed to arise from the constrained binding of prothrombin to prothrombinase. The alternate proposal is that two interconverting forms of the enzyme differentially recognize and cleave the two sites in prothrombin. We have investigated substrate binding using prothrombinase assembled with a catalytically inactive recombinant factor Xa (Xa_S195A) in which Ser₁₉₅ was replaced with Ala and a series of recombinant variants of prothrombin. The prothrombin variants included wild type prothrombin (II_WT) containing both cleavage sites, II_Q271 with Arg₂₇₁ replaced with Gln and a single cleavable site at Arg₃₂₀, II_Q320 with Arg₃₂₀ replaced with Gln and a single cleavable site at Arg₂₇₁ and the uncleavable II_QQ containing both Gln substitutions. Titration of Xa_S195A assembled into prothrombinase with increasing concentrations of the fluorescent active site probe p-aminobenzamidine (PAB) produced a saturable increase in fluorescence confirming the expected ability of Xa_S195A to bind ligands, such as PAB, at the active site despite its lack of catalytic activity. The addition of a saturating concentration of II_WT to reaction mixtures containing PAB and prothrombinase assembled with Xa_S195A resulted in a decrease in PAB fluorescence arising from the engagement of the active site by the substrate and the associated displacement of PAB. Only a minor change in PAB fluorescence was observed with II_QQ even though this uncleavable derivative binds to prothrombinase with the same affinity as II_WT. Thus, features of the substrate-enzyme interaction that determine affinity are independent of active site engagement by the substrate. Saturating concentrations of II_Q271 displaced PAB from the active site of Xa_S195A within prothrombinase to the same extent observed with II_WT while essentially no fluorescence decrease was observed with II_Q320. It follows that although all prothrombin derivatives bind with similar affinity to prothrombinase, elements flanking Arg₃₂₀ can readily engage the active site of Xa_S195A within the enzyme complex while those flanking the Arg₂₇₁ site cannot. Our equilibrium binding measurements establish a primary role for active site binding in determining the perceived rate constant for catalysis without influencing substrate affinity. The differential action of prothrombinase on the two cleavage sites in prothrombin likely arises from constraints imposed by active site-independent interactions that drive substrate affinity and permit active site docking by the Arg₃₂₀ site in the substrate but not the Arg₂₇₁ site. These constraints in active site engagement explain asymmetry in the recognition of the two bonds in intact prothrombin and its ordered cleavage by prothrombinase.