Department of Microbial Biotechnology, Institute of Biochemistry and Biotechnology, Faculty of Life Sciences, Martin-Luther-University Halle-Wittenberg, D-06120 Halle/Saale, Germany.
J Biol Chem. 2010 Apr 30;285(18):13685-93. doi: 10.1074/jbc.M109.082206. Epub 2010 Mar 1.
The RNA-dependent RNA polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B was confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded RNA with high affinity (nanomolar range) and in a stepwise process that reflects the substrate positioning. As demonstrated by CD, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.
RNA 依赖性 RNA 聚合酶 NS5B 是丙型肝炎病毒 (HCV) 复制的关键酶，也是主要的治疗靶点。本研究应用新型连续分析方法，使用高度纯化的蛋白和荧光 RNA 模板，首次全面描述了酶反应的机制。通过荧光光谱法，证实 NS5B 的动力学由两个半反应组成，即底物结合和周转率。确定底物的结合常数和各个反应步骤的速率常数后，表明 NS5B 以高亲和力（纳摩尔范围）结合单链 RNA 模板，并以反映底物定位的逐步过程进行。如 CD 所示，NTP(s) 结合导致酶的三级结构发生变化，形成活性构象。第二个半反应被剖析为顺序聚合和随后的限速产物释放反应。利用这些工具，我们分析了 NS5B 抑制剂 HCV-796 的作用机制，该抑制剂通过阻断第二个半反应来干扰双链 RNA 的形成。
