Cook R R, McRae B J, Powers J C
Arch Biochem Biophys. 1984 Oct;234(1):82-8. doi: 10.1016/0003-9861(84)90326-6.
Several peptide thioester substrates have been synthesized and tested with human thrombins (alpha, gamma, and nitrated), bovine thrombin, trypsin, and Factor X alpha beta. The substrates include various thioalkyl esters, thiobenzyl esters with substitution in the 4-position, substrates containing additional residues on the amino-terminal side of the scissile bond (P extended substrates) and one substrate containing additional residues on the carboxyl-terminal side (P' extended substrate). Neither the P nor the P' extensions resulted in significantly increased specificity; however, with one P extended substrate, D-Phe-Pro-Arg-SBzl, the KM with bovine thrombin (0.72 microM) was the second lowest KM yet reported. The results of this study underscore the importance of P vs. P' extension for thrombin substrates. The kinetic constants of the thiobenzyl esters were found to be little affected by the 4-position substitutions. A comparison of gamma-thrombin and nitrothrombin shows them to be quite similar kinetically, while both are significantly less reactive than alpha-thrombin. With these substrates, trypsin, bovine thrombin, and Factor X alpha beta have kcat/KM values in the approximate ratio of 35:10:1, respectively. The results presented here should be of value in the future design of reactive yet specific substrates for thrombin. The comparisons between the various enzymes could be helpful in clarifying the nature of their active sites.
已经合成了几种肽硫酯底物，并用人凝血酶（α、γ和硝化的）、牛凝血酶、胰蛋白酶和因子Xαβ进行了测试。这些底物包括各种硫代烷基酯、在4位有取代基的硫代苄酯、在裂解键氨基末端侧含有额外残基的底物（P扩展底物）以及一种在羧基末端侧含有额外残基的底物（P'扩展底物）。P扩展和P'扩展均未导致特异性显著提高；然而，对于一种P扩展底物D-Phe-Pro-Arg-SBzl，其与牛凝血酶的米氏常数（KM）为0.72 microM，是迄今报道的第二低的KM。本研究结果强调了凝血酶底物P扩展与P'扩展的重要性。发现硫代苄酯的动力学常数受4位取代的影响很小。γ-凝血酶和硝化凝血酶的比较表明它们在动力学上非常相似，而两者的反应活性均明显低于α-凝血酶。对于这些底物，胰蛋白酶、牛凝血酶和因子Xαβ的催化常数与米氏常数之比（kcat/KM）分别约为35:10:1。本文给出的结果对于未来设计具有反应活性且特异性的凝血酶底物应具有价值。各种酶之间的比较可能有助于阐明其活性位点的性质。
