Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.
Prothrombinase catalyzes thrombin formation by the ordered cleavage of two peptide bonds in prothrombin. Although these bonds are likely approximately 36 A apart, sequential cleavage of prothrombin at Arg-320 to produce meizothrombin, followed by its cleavage at Arg-271, are both accomplished by equivalent exosite interactions that tether each substrate to the enzyme and facilitate presentation of the scissile bond to the active site of the catalyst. We show that impairing the conformational transition from zymogen to active proteinase that accompanies the formation of meizothrombin has no effect on initial cleavage at Arg-320 but inhibits subsequent cleavage at Arg-271. Full thermodynamic rescue of this defective mutant was achieved by stabilizing the proteinase-like conformation of the intermediate with a reversible, active site-specific inhibitor. Irreversible stabilization of intact prothrombin in a proteinase-like state, even without prior cleavage at Arg-320, also enhanced cleavage at Arg-271. Our results indicate that the sequential presentation and cleavage of the two scissile bonds in prothrombin activation is accomplished by substrate bound either in the zymogen or proteinase conformations. The ordered cleavage of prothrombin by prothrombinase is driven by ratcheting of the substrate from the zymogen to the proteinase-like states.
Bianchini
Elsa P
EP
Joseph Stokes Research Institute, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
Orcutt
Steven J
SJ
Panizzi
Peter
P
Bock
Paul E
PE
Krishnaswamy
Sriram
S
D001665:Binding Sites; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009564:Nitracrine / Q000031:analogs & derivatives; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
D004792:Enzyme Precursors; C024890:C 137; D009564:Nitracrine; D011516:Prothrombin; D013925:Thromboplastin; D013917:Thrombin; C026812:meizothrombin