The steady-state kinetic parameters of human α-thrombin and the α-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°C, the Km values for α-thrombin and the complex for S-2238 were 7.9 μm and 7.7 μm, 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 α-thrombin. This difference in the kinetic parameter between α-thrombin and the complex was also observed using the fluorogenic substrate, Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. Morever, the fibrinogen clotting activity of the a-thrombin-staphyloco-agulase complex was less than half that of a-thrombin, suggesting that the α-thrombin active site in the complex is different in catalytic ability from that of free α-thrombin. Other evidence supporting this view was as follows: (1) The α-thrombin-staphyloco-agulase complex is insensitive to antithrombin III, (2) the complex shows much weaker binding to hirudin, as compared to free α-thrombin, and (3) the amidase pH-profiles of the complex and free α-thrombin differ from each other. These results indicate that the microenvironment of the active site of α-thrombin is significantly altered by the complex formation with staphylocoagulase.
|Number of pages||6|
|Journal||Journal of Biochemistry|
|Publication status||Published - Jan 1 1985|
All Science Journal Classification (ASJC) codes
- Molecular Biology