Kawabata S, Morita T, Iwanaga S, Igarashi H
J Biochem. 1985 Apr;97(4):1073-8. doi: 10.1093/oxfordjournals.jbchem.a135150.
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.
测定了人α-凝血酶和α-凝血酶-葡萄球菌凝固酶复合物针对生色底物H-D-苯丙氨酸-哌啶-精氨酸-对硝基苯胺（S-2238）的稳态动力学参数。在pH 8.0和37℃条件下，α-凝血酶和该复合物对S-2238的Km值分别为7.9μM和7.7μM。该复合物催化的这种酰胺酶反应的kcat为127 s-1，明显低于游离α-凝血酶测定的197 s-1的kcat。使用荧光底物Boc-缬氨酸-脯氨酸-精氨酸-4-甲基香豆素-7-酰胺时，也观察到α-凝血酶和该复合物之间动力学参数的这种差异。此外，α-凝血酶-葡萄球菌凝固酶复合物的纤维蛋白原凝血活性不到α-凝血酶的一半，这表明复合物中的α-凝血酶活性位点在催化能力上与游离α-凝血酶不同。支持这一观点的其他证据如下：α-凝血酶-葡萄球菌凝固酶复合物对抗凝血酶III不敏感，与游离α-凝血酶相比，该复合物与水蛭素的结合弱得多，并且该复合物和游离α-凝血酶的酰胺酶pH曲线彼此不同。这些结果表明，与葡萄球菌凝固酶形成复合物会显著改变α-凝血酶活性位点的微环境。
