Binding of substrate in two conformations to human prothrombinase drives consecutive cleavage at two sites in prothrombin

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (IIWT) contained both cleavage sites; IIQ271 contained a single cleavable site at Arg(320); IIQ320 and IIA320 contained a single cleavable site at Arg(271); and IIQQ was resistant to cleavage. Cleavage at Arg(320) in IIQ271 could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or IIWT, whereas cleavage at Arg(271) in either IIQ320 or IIA320 was found to be similar to30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an similar to 30-fold reduction in V-max. Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. IIQ320 was a competitive inhibitor of IIQ271 cleavage, and IIQQ was a competitive inhibitor for each reaction with K-i approximate to K-m. The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.