Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate
Inhibition of a metal-dependent viral RNA triphosphatase by decavanadate
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DOI：
10.1042/bj20060198
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发表时间：
2006-09-15
影响因子：
4.1
通讯作者：
Bisaillon, Martin
中科院分区：
文献类型：
--
作者：
Bougie, Isabelle;Bisaillon, Martin
草履虫囊藻病毒是一种在单细胞小球藻样藻类中复制的大型DNA病毒，编码RNA三磷酸酶，其参与在病毒mRNA的5'端发现的RNA帽结构的合成。小球藻病毒RNA三磷酸酶是金属依赖性RNA三磷酸酶中最小的成员，包括来自真菌、DNA病毒、原生动物和微孢子虫寄生虫的酶。在本研究中，我们研究了各种钒酸含氧阴离子抑制磷酸水解酶活性的能力。荧光光谱和CD研究用于直接监测十钒酸盐与酶的结合。此外，竞争试验表明，decavanadate是一种有效的磷酸水解酶活性的非竞争性抑制剂，诱变研究表明，decavanadate的结合不涉及位于酶的活性位点的氨基酸。为了提供额外的洞察酶的结构和decavanadate结合之间的关系，我们相关的decavanadate结合蛋白质结构的影响，使用CD和胍氯化物诱导的变性作为结构指标。我们的数据表明，在十钒酸盐结合后，整体蛋白质结构没有发生显著的改变。然而，荧光光谱和CD实验清楚地表明，decavanadate的酶的结合显着降低了酶的结构稳定性。总之，这些研究提供了至关重要的见解，金属依赖性RNA三磷酸酶的抑制十钒酸盐。
Paramecium bursaria chlorella virus, a large DNA virus that replicates in unicellular Chlorella-like algae, encodes an RNA triphosphatase which is involved in the synthesis of the RNA cap structure found at the 5' end of the viral mRNAs. The Chlorella virus RNA triphosphatase is the smallest member of the metal-dependent RNA triphosphatases that include enzymes from fungi, DNA viruses, protozoans and microsporidian parasites. In the present study, we investigated the ability of various vanadate oxoanions to inhibit the phosphohydrolase activity of the enzyme. Fluorescence spectroscopy and CD studies were used to directly monitor the binding of decavanadate to the enzyme. Moreover, competition assays show that decavanadate is a potent non-competitive inhibitor of the phosphohydrolase activity, and mutagenesis studies indicate that the binding of decavanadate does not involve amino acids located in the active site of the enzyme. In order to provide additional insight into the relationship between the enzyme structure and decavanadate binding, we correlated the effect of decavanadate binding on protein structure using both CD and guanidinium chloride-induced denaturation as structural indicators. Our data indicated that no significant modification of the overall protein architecture was occurring upon decavanadate binding. However, both fluorescence spectroscopy and CD experiments clearly revealed that the binding of decavanadate to the enzyme significantly decreased the structural stability of the enzyme. Taken together, these studies provide crucial insights into the inhibition of metal-dependent RNA triphosphatases by decavanadate.