Analysis of solvent nucleophile isotope effects: Evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis
Analysis of solvent nucleophile isotope effects: Evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis
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DOI：
10.1021/bi049188f
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发表时间：
2004-08-17
期刊：
影响因子：
2.9
通讯作者：
Harris, ME
中科院分区：
文献类型：
--
作者：
Cassano, AG;Anderson, VE;Harris, ME
重原子同位素效应是研究化学和酶促反应机理的重要工具；然而，它们并没有广泛应用于研究亲核活化的机制。我们开发了分析溶剂O-18亲核同位素效应(（18)k(nuc)）的方法，首次将其应用于核苷酸和核酸的水解反应。在这里，我们报道了由Mg2+和Mg2+依赖的RNase P核酶催化的磷酸二酯水解和由Zn2+依赖的蛋白酶腺苷脱氨酶（ADA）脱氨的（18）k（nuc）。由于ADA将单个溶剂分子掺入产物肌苷中，因此该反应可用于监测复杂反应混合物中溶剂O-18/O-16的比例。该方法与全分子质谱分析磷酸核苷酸同位素比率的新方法相结合，允许测定胸苷5 '-对硝基苯基磷酸水解和RNA裂解的18knuc核糖酶。对于ADA，观察到逆（18）k（nuc）为0.986 +/- 0.001，反映了活性位点Zn2+离子与亲核试剂的配位和逐步机制。相比之下，磷酸二酯反应的（18）k（nuc）是正常的：Mg2+和核酶催化的反应分别为1.027 +/- 0.013和1.030 +/- 0.012。这些正常效应表明，亲核攻击发生在这些反应的限速步骤中，与协调一致的机制一致。然而，这些数值明显小于氢氧根亲核攻击时的（18）k（nuc）值（1.0158 +/- 0.007），表明亲核试剂在过渡态的成键环境更为“僵硬”。动力学：Mg2+催化反应的分析表明，Mg2+-氢氧化物配合物是催化物质；因此，较低的（18）k（nuc）在很大程度上反映了亲核氧的直接金属离子配位。RNase P核酶催化反应的类似值为金属离子催化的亲核活化提供了支持。
Heavy atom isotope effects are a valuable tool for probing chemical and enzymatic reaction mechanisms; yet, they are not widely applied to examine mechanisms of nucleophilic activation. We developed approaches for analyzing solvent O-18 nucleophile isotope effects ((18)k(nuc)) that allow, for the first time, their application to hydrolysis reactions of nucleotides and nucleic acids. Here, we report (18)k(nuc) for phosphodiester hydrolysis catalyzed by Mg2+ and by the Mg2+-dependent RNase P ribozyme and deamination by the Zn2+-dependent protein enzyme adenosine deaminase (ADA). Because ADA incorporates a single solvent molecule into the product inosine, this reaction can be used to monitor solvent O-18/O-16 ratios in complex reaction mixtures. This approach, combined with new methods for analysis of isotope ratios of nucleotide phosphates by whole molecule mass spectrometry, permitted determination of 18knuc for hydrolysis of thymidine 5'-p-nitrophenyl phosphate and RNA cleavage by the RNase P ribozyme. For ADA, an inverse (18)k(nuc) of 0.986 +/- 0.001 is observed, reflecting coordination of the nucleophile by an active site Zn2+ ion and a stepwise mechanism. In contrast, the observed (18)k(nuc) for phosphodiester reactions were normal: 1.027 +/- 0.013 and 1.030 +/- 0.012 for the Mg2+-and ribozyme-catalyzed reactions, respectively. Such normal effects indicate that nucleophilic attack occurs in the rate-limiting step for these reactions, consistent with concerted mechanisms. However, these magnitudes are significantly less than the (18)k(nuc) observed for nucleophilic attack by hydroxide (1.0158 +/- 0.007), indicating a "stiffer" bonding environment for the nucleophile in the transition state. Kinetic: analysis of the Mg2+-catalyzed reaction indicates that a Mg2+-hydroxide complex is the catalytic species; thus, the lower (18)k(nuc) in large part, reflects direct metal ion coordination of the nucleophilic oxygen. A similar value for the RNase P ribozyme catalyzed reaction provides support for nucleophilic activation by metal ion catalysis.