Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.
Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.
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DOI：
10.1021/acs.biochem.1c00489
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发表时间：
2021-09-21
期刊：
影响因子：
2.9
通讯作者：
Harris, Michael E.
中科院分区：
文献类型：
--
作者：
Yoon, Suhyun;Harris, Michael E.
酸/碱催化是核糖核酸酶和核酶使用的重要催化策略;然而，理解参与质子转移的官能团的数量和身份仍然具有挑战性。质子库存（PI）技术分析的依赖性的酶反应速率上的D2 O到H2O的比例，并可以提供有关的可交换的网站，产生同位素效应的数量和它们的大小的信息。Gross-Butler（GB）方程用于从通常在以下条件下收集的PI数据评估H/D分馏因子（即，pH-速率曲线中的“平台”），假定活性位点残基电离的变化最小。然而，限制PI分析这些条件是有问题的许多核糖核酸酶，核酶，和它们的变体，由于在活性位点残基的作用的模糊性，缺乏一个平台内可访问的pL范围内，或进行电离的活性位点官能团之间的合作相互作用。在这里，我们扩展到GB方程，首先使用的贝维拉夸和同事的HDV核酶的非合作模型中的替代酶状态的物种分布的整合，开发一个通用的人口加权GB方程，允许模拟和全球拟合的三维关系的D2 O比（n）与pL与kn/k 0。使用GPW-GB方程对RNase A、HDVrz和VSrz的PI结果进行模拟，表明在pL速率曲线中以多个选定的pL值获得的数据可以帮助规划和解释溶剂同位素效应实验，以区分替代机制模型。
Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross–Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a “plateau” in the pH–rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn/k0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL–rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.
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