Detailed structural knowledge of enzyme-inhibitor complexes trapped in intermediate state is the key for a fundamental understanding of reaction mechanisms taking place in enzymes and is indispensable as a structure-guided drug design tool. Solution state NMR uniquely allows the study of active sites of enzymes in equilibrium between different tautomeric forms. In this study 1H, 19F and 15 N NMR spectroscopy has been used to probe the interaction contacts of inhibitors locked in transition states of the catalytic triad of a serine protease. It was demonstrated on the serotype II Dengue virus NS2B:NS3pro serine protease and its mutants, H51N and S135A, in complex with high-affinity ligands containing trifluoromethyl ketone (tfk) and boronic groups in the C-terminal of tetra-peptides. Monitoring 19F resonances, shows that only one of the two isomers of the tfk tetra-peptide binds with NS2B:NS3pro and that access to the bulk of the active site is limited. Moreover, there were no bound water found in proximity of the active site for any of the ligands manifesting in a favorable condition for formation of low barrier hydrogen bonds (LBHB) in the catalytic triad. Based on this data we were able to identify a locked conformation of the protein active site. The data also indicates that the different parts of the binding site most likely act independently of each other. Our reported findings increases the knowledge of the detailed function of the catalytic triad in serine proteases and could facilitate the development of rational structure based inhibitors that can selectively target the NS3 protease of Dengue type II (DENV2) virus. In addition the results shows the usefulness of probing active sites using 19F NMR spectroscopy.

中文翻译：

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通过1H，19F和15 N NMR光谱探测DENV2型丝氨酸蛋白酶及其突变体催化三联体中锁定在过渡态的抑制剂的接触。

捕获处于中间状态的酶抑制剂复合物的详细结构知识是对酶中发生的反应机理有基本了解的关键，并且作为结构指导的药物设计工具必不可少。溶液状态NMR独特地允许研究不同互变异构形式之间平衡中酶的活性位点。在这项研究中，已使用1H，19F和15 N NMR光谱探测锁定在丝氨酸蛋白酶催化三联体过渡态的抑制剂的相互作用接触。在血清型II型登革热病毒NS2B：NS3pro丝氨酸蛋白酶及其突变体H51N和S135A上与在四肽C端含有三氟甲基酮（tfk）和硼基的高亲和力配体配合使用证明了这一点。监视19F共振，结果表明，tfk四肽的两个异构体中只有一个与NS2B：NS3pro结合，并且对大部分活性位点的访问受到限制。而且，对于在催化三联体中形成低阻挡氢键（LBHB）的有利条件显示的任何配体，在活性位点附近都没有发现结合水。基于这些数据，我们能够鉴定出蛋白质活性位点的锁定构象。数据还表明结合位点的不同部分最有可能彼此独立地起作用。我们报告的发现增加了对催化三联体在丝氨酸蛋白酶中详细功能的了解，并且可以促进基于理性结构的抑制剂的发展，该抑制剂可以选择性地靶向登革热II型（NSV2）病毒的NS3蛋白酶。