Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 1073-1078 |
| Number of pages | 6 |
| Journal | Journal of Biochemistry |
| Volume | 97 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - Jan 1 1985 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
Cite this
Difference in enzymatic properties between α-thrombin-staphylocoagulase complex and free α-thrombin. / Kawabata, Shun-Ichiro; Morita, Takashi; Iwanaga, Sadaaki; Igarashi, Hideo.
In: Journal of Biochemistry, Vol. 97, No. 4, 01.01.1985, p. 1073-1078.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Difference in enzymatic properties between α-thrombin-staphylocoagulase complex and free α-thrombin
AU - Kawabata, Shun-Ichiro
AU - Morita, Takashi
AU - Iwanaga, Sadaaki
AU - Igarashi, Hideo
PY - 1985/1/1
Y1 - 1985/1/1
N2 - 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.
AB - 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.
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U2 - 10.1093/oxfordjournals.jbchem.a135150
DO - 10.1093/oxfordjournals.jbchem.a135150
M3 - Article
C2 - 4030715
AN - SCOPUS:0021987011
VL - 97
SP - 1073
EP - 1078
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 4
ER -