title: Difference in enzymatic properties between alpha-thrombin-staphylocoagulase complex and free alpha-thrombin.
abstract: The steady-state kinetic parameters of human alpha-thrombin and the alpha-thrombin-staphylocoagulase complex as to the chromogenic substrate, H-D-Phe-Pip-Arg-p-nitroanilide (S-2238), were determined. At pH 8.0 and 37 degrees C, the Km values for alpha-thrombin and the complex for S-2238 were 7.9 microM and 7.7 microM, respectively. The kcat of this amidase reaction catalyzed by the complex was 127 s-1, which had apparently decreased from the kcat of 197 s-1 determined for free alpha-thrombin. This difference in the kinetic parameter between alpha-thrombin and the complex was also observed using the fluorogenic substrate, Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. Moreover, the fibrinogen clotting activity of the alpha-thrombin-staphylocoagulase complex was less than half that of alpha-thrombin, suggesting that the alpha-thrombin active site in the complex is different in catalytic ability from that of free alpha-thrombin. Other evidence supporting this view was as follows: The alpha-thrombin-staphylocoagulase complex is insensitive to antithrombin III, the complex shows much weaker binding to hirudin, as compared to free alpha-thrombin, and the amidase pH-profiles of the complex and free alpha-thrombin differ from each other. These results indicate that the microenvironment of the active site of alpha-thrombin is significantly altered by the complex formation with staphylocoagulase.
mesh_terms: D000990:Antithrombin III / Q000494:pharmacology; D003030:Coagulase / Q000378:metabolism*; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000378:metabolism*
chemical_list: D003030:Coagulase; D006629:Hirudins; D046911:Macromolecular Substances; D000990:Antithrombin III; D013917:Thrombin