Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin

摘要: Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action prothrombinase on Arg(320) followed by is implied intermediate observed during activation. We have studied individual reactions catalyzed using a series recombinant derivatives: wild type (II(WT)) contained both sites; II(Q271) single cleavable site at Arg(320); II(Q320) II(A320) Arg(271); II(QQ) was resistant to cleavage. Cleavage in could account initial reaction leading consumption either plasma or II(WT), whereas found be approximately 30-fold slower. Equivalent kinetic constants were obtained three four possible half-reactions. Slow intact resulted an reduction V(max). Thus, pathway bond can explained reactions. competitive inhibitor each with K(i) K(m). The data inconsistent previous proposals suggest model which substrates half-reactions bind mutually exclusive manner equal affinity site-independent way. Despite equivalent exosite binding interactions between all enzyme, we propose that ordered constraints associated one two conformations form prothrombinase.