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

• Published in: The Journal of biological chemistry 2004-12, Vol.279 (52), p.54927-54936
• Main Authors: Orcutt, Steven J., Krishnaswamy, Sriram
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arginine - metabolism; Binding Sites; Cell Line; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Mutagenesis, Insertional; Peptide Fragments - chemistry; Protein Binding; Protein Conformation; Prothrombin - chemistry; Prothrombin - genetics; Prothrombin - metabolism; Recombinant Proteins - metabolism; Structure-Activity Relationship; Substrate Specificity; Thromboplastin - chemistry; Thromboplastin - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M410866200

Thrombin formation results from cleavage of prothrombin following Arg271 and Arg320. Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg320 followed by Arg271 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 Arg320; IIQ320 and IIA320 contained a single cleavable site at Arg271; and IIQQ was resistant to cleavage. Cleavage at Arg320 in IIQ271 could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or IIWT, whereas cleavage at Arg271 in either IIQ320 or IIA320 was found to be ∼30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg271 in intact prothrombin resulted from an ∼30-fold reduction in Vmax. 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 Ki ≈ Km. 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.