|A list of the prothrombin variants used to demonstrate the importance of geometric positioning in prothrombinase activity.
The conversion of prothrombin to thrombin is crucial for proper clot formation during wound repair. This conversion is handled by the membrane-bound prothrombinase complex, which contains a protease that sequentially cleaves prothrombin at Arg320, followed by Arg271. This defined chain of events is believed to occur through exosite-dependent binding of prothrombin to prothrombinase, arranging the substrate to precisely present Arg320 in the protease active site. The recent discovery of a reduced-activity prothrombin with a G319R mutation questions the necessity of precise geometry. In this study, the researchers systematically assessed the importance of substrate positioning. They used a series of cleavage site variants that incrementally shifted the Arg320 site toward the Arg271 site. Functionally, they observed that prothrombinase was quite tolerant of N-terminal shifts of the cleavage site by one or two residues, although larger shifts led to an abrupt loss in activity. In contrast, prothrombin docking was strongly dependent on the position and sequence of the scissile bond segment, and even minor perturbations resulted in strong thermodynamic increases.
Regulated Cleavage of Prothrombin by Prothrombinase. Repositioning a Cleavage Site Reveals the Unique Kinetic Behavior of the Action of Prothrombinase on Its Compound Substrate
Harlan N. Bradford, Joseph A. Micucci and Sriram Krishnaswamy
J. Biol. Chem., published online Oct. 26, 2009