Complexity in pH-Dependent Ribozyme Kinetics: Dark p&ITK&ITa Shifts and Wavy Rate-pH Profiles
Complexity in pH-Dependent Ribozyme Kinetics: Dark p&ITK&ITa Shifts and Wavy Rate-pH Profiles
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DOI：
10.1021/acs.biochem.7b00784
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发表时间：
2018-02-06
期刊：
影响因子：
2.9
通讯作者：
Bevilacqua, Philip C.
中科院分区：
文献类型：
--
作者：
Frankel, Erica A.;Bevilacqua, Philip C.
带电碱基存在于 RNA 酶或核酶中，它们在催化中发挥关键作用。阳离子碱提供质子并进行静电催化，而阴离子碱接受质子。我们之前发表了根据一般酸和一般碱的物种图对核酶的速率-pH 曲线进行的模拟，这对于理解核酶如何响应 pH 非常有用。在那项研究中，我们没有考虑RNA上一般酸和一般碱之间的相互作用或与其他物种的相互作用。自该报告发布以来，人们发现了多种小型核酶，其中许多在活性位点带有带电核碱基或金属离子，它们可以直接相互作用并参与催化，也可以作为“影响者”间接相互作用。在此，我们根据反向质子化带电核碱基相互作用的物种图模拟了实验速率-pH 曲线。这些分析揭示了 pH 依赖性酶动力学的两个令人惊讶的特征。 (1) 一般酸和一般碱之间的协同作用，增强核酶功能形式的数量，并表现为隐藏或“暗”pK(a) 变化，真正的 pK(a) 变化加速反应，但不易通过标准实验方法观察到，(2) 影响因素有利地改变质子转移核碱基的 pK(a)，并表现为“波状”速率-pH 曲线。我们发现了与蛋白酶文献的相似之处，包括反质子化和波状行为，同时指出 RNA 更容易发生反质子化。由简单的成对相互作用产生的复杂性，应有助于对 RNA 和蛋白酶的复杂速率-pH 曲线进行解卷积，并提出可通过实验和计算进行测试的用于促进催化作用的隐蔽催化装置。
Charged bases occur in RNA enzymes, or ribozymes, where they play key roles in catalysis. Cationic bases donate protons and perform electrostatic catalysis, while anionic bases accept protons. We previously published simulations of rate-pH profiles for ribozymes in terms of species plots for the general acid and general base that have been useful for understanding how ribozymes respond to pH. In that study, we did not consider interaction between the general acid and general base or interaction with other species on the RNA. Since that report, diverse small ribozyme classes have been discovered, many of which have charged nucleobases or metal ions in the active site that can either directly interact and participate in catalysis or indirectly interact as "influencers". Herein, we simulate experimental rate-pH profiles in terms of species plots in which reverse protonated charged nucleobases interact. These analyses uncover two surprising features of pH-dependent enzyme kinetics. (1) Cooperativity between the general acid and general base, enhances population of the functional forms of a ribozyme and manifests itself as hidden or "dark" pK(a) shifts, real pK(a) shifts that accelerate the reaction but are not readily observed by standard experimental approaches, and (2) influencers favorably shift the pK(a)s of proton-transferring nucleobases and manifest themselves as "wavy" rate-pH profiles. We identify parallels with the protein enzyme literature, including reverse protonation and wavelike behavior, while pointing out that RNA is more prone to reverse protonation. The, complexities uncovered, which arise from simple pairwise interactions, should aid deconvolution of complex rate-pH profiles for RNA and protein enzymes and suggest veiled catalytic devices for promoting catalysis that can be tested by experiment and calculation.