Effects of Substrate Geometry on Active Site Engagement Govern the Preferential Action of Prothrombinase on One of the Two Cleavage Sites in Prothrombin.

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 (II_WT) with D-R-R³²⁰ (II_RR) or R-G-R³²⁰ (II_RGR) yielded substrate species that were indistinguishable from II_WT 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 II_WT with Q (II_Q320) 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 II_Q320 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 II_WT. For either variant, the rate of prothrombin consumption was within 2-fold that observed with II_WT 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 II_Q320. 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 Xa_A195. 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.