Long-Range Interactions in the Alpha Subunit of Tryptophan Synthase Help to Coordinate Ligand Binding, Catalysis, and Substrate Channeling
Long-Range Interactions in the Alpha Subunit of Tryptophan Synthase Help to Coordinate Ligand Binding, Catalysis, and Substrate Channeling
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DOI：
10.1016/j.jmb.2013.01.030
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发表时间：
2013-05-13
影响因子：
5.6
通讯作者：
Boehr, David D.
中科院分区：
文献类型：
--
作者：
Axe, Jennifer M.;Boehr, David D.
色氨酸合酶的α-亚基（α TS）催化吲哚-3-甘油磷酸转化为D甘油醛-3-磷酸和吲哚。我们建议，变构网络固有的α TS调制的β-亚基的结合，以调节α TS功能。因此，了解α TS中的这些长程氨基酸网络可以深入了解TS中两个活性位点的协调。在这项研究中，我们已经使用Ala残基作为探针的结构和动态变化的aTS在整个催化循环中，在β-亚基的情况下。通过位点特异性氨基酸取代和配体滴定的化学位移变化的投影分析表明，α TS具有三种重要的构象状态：无配体、甘油醛-3-磷酸结合（样）和活性状态。化学位移相关分析表明，这些构象中的氨基酸网络是不同的。特别是，有长程连接，只有在活性状态下，之间的Ala 47，它报告的结构和动态变化与一般的酸/碱Glu 49，和残基内的β 2 α 2环，其中包含催化重要的Asp 60残基。这些长程相互作用对于协调化学催化可能是重要的。在自由状态下，而不是在活性状态下，在β 2 α 2和β 6 α 6环之间存在可能有助于协调底物结合的连接。变构网络的变化也伴随着蛋白质的动态变化。在催化周转期间，蛋白质在毫秒时间尺度上变得更加刚性，并且活性位点动力学被驱动到更快的纳秒时间尺度。(C)2013爱思唯尔有限公司版权所有。
The alpha-subunit of tryptophan synthase (alpha TS) catalyzes the conversion of indole-3-glycerol phosphate to Dglyceraldehyde-3-phosphate and indole. We propose that allosteric networks intrinsic to alpha TS are modulated by the binding of the beta-subunit to regulate alpha TS function. Understanding these long-range amino acid networks in alpha TS thus gives insight into the coordination of the two active sites within TS. In this study, we have used Ala residues as probes for structural and dynamic changes of aTS throughout its catalytic cycle, in the absence of the beta-subunit. Projection analysis of the chemical shift changes by site-specific amino acid substitutions and ligand titrations indicates that alpha TS has three important conformational states: ligand-free, glyceraldehyde-3-phosphate-bound(like), and the active states. The amino acid networks within these conformations are different, as suggested by chemical shift correlation analysis. In particular, there are long-range connections, only in the active state, between Ala47, which reports on structural and dynamic changes associated with the general acid/base Glu49, and residues within the beta 2 alpha 2 loop, which contains the catalytically important Asp60 residue. These long-range interactions are likely important for coordinating chemical catalysis. In the free state, but not in the active state, there are connections between the beta 2 alpha 2 and beta 6 alpha 6 loops that likely help to coordinate substrate binding. Changes in the allosteric networks are also accompanied by protein dynamic changes. During catalytic turnover, the protein becomes more rigid on the millisecond timescale and the active-site dynamics are driven to a faster nanosecond timescale. (C) 2013 Elsevier Ltd. All rights reserved.