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 Arg 320 followed by cleavage at Arg 271 . The largely ordered activation arises because Arg 320 in intact prothrombin is hydrolyzed by prothrombinase with a Vmax that is ~30-fold greater than that for cleavage at Arg 271 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 Ser 195 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 Arg 271 replaced with Gln and a single cleavable site at Arg 320 , II Q320 with Arg 320 replaced with Gln and a single cleavable site at Arg 271 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 Arg 320 can readily engage the active site of Xa S195A within the enzyme complex while those flanking the Arg 271 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 Arg 320 site in the substrate but not the Arg 271 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.