Difference in enzymatic properties between α-thrombin-staphylocoagulase complex and free α-thrombin

Shun-Ichiro Kawabata, Takashi Morita, Sadaaki Iwanaga, Hideo Igarashi

研究成果: ジャーナルへの寄稿記事

25 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)1073-1078
ページ数6
ジャーナルJournal of Biochemistry
97
発行部数4
DOI
出版物ステータス出版済み - 1 1 1985

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Coagulase
Thrombin
S 2238
amidase
Kinetic parameters
Catalytic Domain
Hirudins
Chromogenic Compounds
Antithrombin III
Fluorescent Dyes
Fibrinogen

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

これを引用

Difference in enzymatic properties between α-thrombin-staphylocoagulase complex and free α-thrombin. / Kawabata, Shun-Ichiro; Morita, Takashi; Iwanaga, Sadaaki; Igarashi, Hideo.

:: Journal of Biochemistry, 巻 97, 番号 4, 01.01.1985, p. 1073-1078.

研究成果: ジャーナルへの寄稿記事

Kawabata, Shun-Ichiro ; Morita, Takashi ; Iwanaga, Sadaaki ; Igarashi, Hideo. / Difference in enzymatic properties between α-thrombin-staphylocoagulase complex and free α-thrombin. :: Journal of Biochemistry. 1985 ; 巻 97, 番号 4. pp. 1073-1078.
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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.",
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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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