Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid-base catalysis and substrate-dependent metal ion activation.
Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid-base catalysis and substrate-dependent metal ion activation.
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冠状病毒NSP15核酸内切酶RNA裂解的动力学分析：酸碱催化和底物依赖金属离子激活的证据。
DOI：
10.1016/j.jbc.2023.104787
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发表时间：
2023-06
影响因子：
4.8
通讯作者：
Harris, Michael E
中科院分区：
文献类型：
--
作者：
Huang, Tong;Snell, Kimberly C;Kalia, Nidhi;Gardezi, Shahbaz;Guo, Lily;Harris, Michael E
了解严重急性呼吸综合征冠状病毒2型非结构蛋白的功能特性对于确定其在病毒生命周期中的作用，开发改进的治疗和诊断方法以及对抗未来的变体至关重要。冠状病毒非结构蛋白Nsp 15是一种六聚体U特异性内切核酸酶，其功能、底物特异性、机制和动力学尚未完全确定。以前的研究报告说，Nsp 15需要Mn 2+离子的最佳活性;然而，二价离子对Nsp 15反应动力学的影响尚未详细研究。在这里，我们分析了模型ssRNA底物的单次和多次周转动力学。我们的数据证实，二价离子被激活的催化，并显示Mn 2+激活Nsp 15裂解两个不同的ssRNA寡核苷酸底物，但不是一个二核苷酸。ssRNA底物的双相动力学表明，Mn 2+稳定了在酶上具有更快底物裂解的替代酶状态。然而，我们没有检测到Mn 2+诱导的构象变化，使用CD和荧光光谱。在存在和不存在Mn 2+的情况下的pH速率曲线揭示了活性位点可电离基团，其具有相似的约pKas。4.8到5.2。一个Rp立体异构体硫代磷酸酯修改在易裂的磷酸盐催化支持机制，涉及阴离子过渡态的影响最小。然而，SP的立体异构体是无活性的，因为弱结合，与模型的位置nonbridging磷酰氧深的活性位点。总之，这些数据表明，Nsp 15采用传统的酸-碱催化机制，通过阴离子过渡态，二价离子活化是底物依赖性的。
Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid–base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.