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Investigations of the dynamic amino acid networks in the alpha-subunit of tryptophan synthase.

机译：色氨酸合酶α-亚基中动态氨基酸网络的研究。

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摘要

Amino acid networks are a series of noncovalent interactions between residues that can span from surface-exposed residues to those deep in the active site of the protein. These amino acid networks, or 'sectors', have been proposed to be intrinsic to all proteins, and to play a key role in protein allostery. Current methods of identification of networks include computationallybased and experimentally-based methods, though only the latter have the ability to probe amino acid networks under a variety of ligand-bound conditions. A recently developed NMR-based technique is known as the CHEmical Shift Covariance Analysis (CHESCA) method was used to identify and characterize the amino acid networks in isolated alpha-subunits of tryptophan synthase (alphaTS).;Tryptophan synthase (TS) has long been studied for the intersubunit communication observed between the alpha- and beta-subunits (betaTS) as well as the intramolecular channel connecting the two active sites. The conformational states of the two subunits are highly synchronized and it is possible that this extensive intersubunit communication is the result of the amino acid networks connecting alphaTS and betaTS. The use of alphaTS as the system allowed for the study of the enzyme under working conditions (i.e. while undergoing active catalytic turnover) and under resting conditions (i.e. while in the apo state), as well as two other, single ligand-bound states, revealing the ligand-dependency of the alphaTS amino acid networks. Most importantly, two clusters of residues were identified in each state and the catalytic residue, Glu49, switches clusters only in the working state. Additionally, the T183V substitution severs crucial hydrogen bonds between the dynamic beta2alpha2 and beta6alpha6 loops. This disruption appears to prevent the cluster switch of Glu49 and results in a 14-fold decrease in catalytic efficiency. These results suggest that amino acid networks play a crucial role in catalysis and association with the correct cluster is key to enzyme function.
机译：氨基酸网络是残基之间的一系列非共价相互作用，其范围可以从表面暴露的残基到蛋白质活性位点深处的那些残基。已经提出这些氨基酸网络或“区段”是所有蛋白质固有的，并且在蛋白质变构中起关键作用。当前的网络识别方法包括基于计算和基于实验的方法，尽管只有后者具有在各种配体结合条件下探测氨基酸网络的能力。一种新近开发的基于NMR的技术被称为化学位移协方差分析（CHESCA）方法，用于鉴定和表征色氨酸合酶（alphaTS）的分离的α亚基中的氨基酸网络。研究了α-和β-亚基（betaTS）之间以及连接两个活性位点的分子内通道之间的亚基间通讯。这两个亚基的构象状态高度同步，并且这种广泛的亚基间通讯可能是由于氨基酸网络连接alphaTS和betaTS的结果。使用alphaTS作为系统可以研究酶在工作条件下（即进行积极的催化转换）和静止条件下（即在apo态时）以及其他两个单一配体结合状态下的酶，揭示了αTS氨基酸网络的配体依赖性。最重要的是，在每种状态下均鉴定出两个残基簇，而催化残基Glu49仅在工作状态下才转换簇。此外，T183V取代切断了动态beta2alpha2和beta6alpha6环之间的关键氢键。这种破坏似乎阻止了Glu49的簇转换，并导致催化效率降低了14倍。这些结果表明，氨基酸网络在催化中起关键作用，与正确簇的缔合是酶功能的关键。

著录项

  • 作者

    Axe, Jennifer M.;

  • 作者单位

    The Pennsylvania State University.;

  • 授予单位 The Pennsylvania State University.;
  • 学科 Biochemistry.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 193 p.
  • 总页数 193
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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