Prothrombin activation by prothrombinase is a paradigm for proteolytic activation reactions wherein product is produced following cleavage at more than one site in the substrate. Prothrombin is tethered to prothrombinase through exosite binding and is preferentially cleaved at R320. Subsequent cleavage at R271, in the resulting intermediate, yields thrombin. Selective presentation of the R320 site for active site docking and cleavage likely arises from the combined constraints of exosite-dependent tethering and the position of the cleavage site within the polypeptide sequence of prothrombin. A series of recombinant variants with substitutions in residues at and preceding the R320 cleavage site have been employed to investigate the role of geometric effects in enforcing the selective action of prothrombinase on this site in prothrombin. Replacement of the sequence D-G-R320 in wild type prothrombin (IIWT) with D-R-R320 (IIRR) or R-G-R320 (IIRGR) yielded substrate species that were indistinguishable from IIWT in their conversion to thrombin by prothrombinase. Equivalent progress curves for bands produced following initial cleavage at R320 followed by cleavage at R271 were established by SDS-PAGE for all three substrate species. Specific and quantitative cleavage at R320 rather than at R319 or R318 in the initial cleavage reaction was established by N-terminal sequencing. Substitution of R320 in IIWT with Q (IIQ320) rendered this site uncleavable and led to very slow cleavage only at the R271 site. These findings support a role for geometric constraints in precisely positioning R320 in II for preferential cleavage. Surprisingly, substitution of flanking sequences in IIQ320 to yield R-G-R-Q320 (II-1Shift) and R-G-R-G-Q320 (II-2Shift) yielded variants that were cleaved in a manner similar to IIWT. For either variant, the rate of prothrombin consumption was within 2-fold that observed with IIWT and could be quantitatively explained by cleavage at R319 (II-1Shift) or R318 (II-2Shift) determined by N-terminal sequencing. Thus, provided position 320 cannot be cleaved, geometric effects are not absolute and flexibility can be tolerated in the position of the scissile bond presented for preferential active site engagement. Variants containing RGR substitutions further shifted to the -3 and -4 positions were instead cleaved ~30-fold slower but at R271 similar to IIQ320. Dramatic loss of cleavage seen with these further N-terminal shifts establishes the limits of possible flexibility in substrate presentation to the active site. Further insights were provided by binding studies examining the ability of these variants to engage and displace 4-aminobenzamidine from the active site of the catalyst within prothrombinase assembled with XaA195. R320 in prothrombin engaged the active site with an equilibrium constant that was ~600-fold more favorable than R271. The register shift variants showed systematic decreases in their affinities for engaging the active site. Loss of cleavage at R316 in II-4Shift correlated with a markedly reduced affinity for active site docking. Thus, geometric constraints, arising from exosite binding, play an essential role in affecting the thermodynamics of active docking by prothrombin. However, very significant decreases in the equilibrium constant for active site docking of an otherwise highly favorable interaction are evident as only minor changes in rate. Large changes in rate and qualitative differences in cleavage pathway result when the equilibrium constant for cleavage at R316 in II-4Shift approaches that for R271. Our findings provide an explanation for how the action of prothrombinase on prothrombin is regulated by both precise substrate geometry and competition for active site docking between different sites within the substrate. The unexpected relationship between thermodynamics and rate reveals new mechanistic insights into the ability of prothrombinase to discriminate between sites within prothrombin and how thrombin formation is regulated.

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