Hougland James L, Kravchuk Alexander V, Herschlag Daniel, Piccirilli Joseph A
Department of Chemistry, University of Chicago, Illinois, USA.
PLoS Biol. 2005 Sep;3(9):e277. doi: 10.1371/journal.pbio.0030277. Epub 2005 Aug 16.
The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.
生命系统的生存能力与催化磷酸基转移反应的酶紧密相关。对于这类中的许多酶，包括几种核酶，二价金属离子作为必需的辅助因子。要理解金属离子如何介导催化作用，需要阐明金属离子与酶及底物之间的相互作用。在嗜热四膜虫I组内含子中，先前利用原子诱变和金属离子拯救行为的定量分析的研究确定了三种金属离子（MA、MB和MC），它们在反应过渡态与核酶底物形成五种相互作用。在此，我们将底物原子诱变与核酶骨架中的位点特异性硫代磷酸酯取代相结合，以开发一种强大的通用策略来确定RNA中催化金属离子的配体。在将此策略应用于嗜热四膜虫I组内含子时，我们已确定核苷酸C262处的前手性磷酰氧作为MC的核酶配体。我们的发现建立了核酶核心与化学过渡态功能定义模型之间的直接联系，从而扩展了已知的过渡态相互作用集，并为应用最近的I组内含子晶体结构来理解催化作用提供了关键信息。
