Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases
Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases
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DOI：
10.1016/j.ymeth.2007.09.007
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发表时间：
2008-02-01
期刊：
影响因子：
4.8
通讯作者：
Hou, Ya-Ming
中科院分区：
文献类型：
--
作者：
Francklyn, Christopher S.;First, Eric A.;Hou, Ya-Ming
蛋白质合成的准确性依赖于氨基酰-tRNA合成酶(AARs)区分真实和接近同源底物的能力。到目前为止，对AARSs功能的分析，包括鉴定参与氨基酸的AARs残基和识别tRNA，在很大程度上依赖于稳态动态焦磷酸交换和氨酰化试验。研究一组更有限的AARS系统的稳态前动力学研究也被用来评估单个酶-底物相互作用的能量贡献，特别是在腺化半反应中。最近，人们对使用AARS的快速动力学方法重新产生了兴趣，导致它们被应用到几个新的AARS系统中，从而识别出区分两个结构上不同的AARS类别的机制差异。在这里，我们回顾了AARS函数的热力学和动力学分析技术。在简要介绍了材料的制备方法和稳态动力学分析方法之后，本文将介绍利用快速猝灭和停流荧光分析活化和氨基酰基转移反应的稳态前动力学方法。将这些方法应用到任何AARS系统中，研究人员都可以推导出激活和氨基酰基转移反应的详细动力学机制，从而能够以严格和定量的方式解决底物专一性、立体化学机制和抑制剂相互作用等问题。(C)2007 Elsevier Inc.保留所有权利。
The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and ammoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion. (C) 2007 Elsevier Inc. All rights reserved.