Molecular docking of Subtilisin K2, a fibrin-degrading enzyme from Indonesian moromi, with its substrates

Abstract Fibrinogen supplies the primary building block of the blood clot or thrombus after α-thrombin converts fibrinogen to fibrin during the final phases of coagulation. When the homeostasis system is disrupted, blood clots that aggregate in the blood vessels can lead to thrombosis. Fibrin-degrading enzyme from Bacillus subtilis K2 (Subtilisin K2) of Indonesian moromi has many excellent characteristics apart from its strong fibrinolytic activity. Bioinformatic analysis using the CDART webserver indicated that the enzyme appeared to share a conserved domain with the peptidase s8 superfamily also known as the subtilase family. This study used molecular docking between these fibrin-degrading enzymes and specific substrates (fibrin and fibrinogen) using the HADDOCK webserver and aimed to predict the enzyme mechanism of action. This analysis revealed that the enzyme interlocked with the two substrates; however, it suggested no productive interactions between Subtilisin K2 and fibrinogen. A hydrolysis reaction is suggested between Subtilisin K2 and the fibrin substrate. There was a strong indication that amino acids Asp19, His51, and Ser208 in Subtilisin K2’s active site interacts with Leu168, Ile171, and Leu172 of the fibrin substrate with a ∆G of -19.4 kcal/mol. Subtilisin K2 tends to act more as a fibrin-degrading enzyme than as a fibrinogen-degrading enzyme.

摘要 纤维蛋白原（Fibrinogen）是血凝块或血栓（thrombus）的核心构成原料，在凝血终末期阶段，α-凝血酶（α-thrombin）可将纤维蛋白原转化为纤维蛋白。当机体稳态系统失衡时，在血管内聚集的血凝块可引发血栓形成。源自印尼醪液（Indonesian moromi）的枯草芽孢杆菌K2（Bacillus subtilis K2）所分泌的纤维蛋白降解酶——枯草杆菌蛋白酶K2（Subtilisin K2），除具备极强的纤溶活性外，还拥有诸多优良特性。借助CDART网络服务器（CDART webserver）开展的生物信息学分析显示，该酶与S8家族肽酶（peptidase s8 superfamily，又称枯草杆菌蛋白酶家族subtilase family）共享保守结构域。本研究依托HADDOCK网络服务器（HADDOCK webserver），对该纤维蛋白降解酶与特异性底物（纤维蛋白及纤维蛋白原）进行分子对接（molecular docking）实验，旨在解析该酶的作用机制。分析结果显示，该酶可与两种底物结合，但提示枯草杆菌蛋白酶K2与纤维蛋白原之间未形成有效相互作用；而该酶与纤维蛋白底物之间则存在水解反应。进一步分析证实，枯草杆菌蛋白酶K2活性位点中的天冬氨酸19（Asp19）、组氨酸51（His51）与丝氨酸208（Ser208），可与纤维蛋白底物的亮氨酸168（Leu168）、异亮氨酸171（Ile171）及亮氨酸172（Leu172）发生相互作用，其结合吉布斯自由能变化（∆G）为-19.4千卡每摩尔（kcal/mol）。综上，枯草杆菌蛋白酶K2更倾向于作为纤维蛋白降解酶发挥功能，而非纤维蛋白原降解酶。