Investigation of allosteric linkages in the regulation of tryptophan synthase: The roles of salt bridges and monovalent cations probed by site-directed mutation, optical spectroscopy, and kinetics
Investigation of allosteric linkages in the regulation of tryptophan synthase: The roles of salt bridges and monovalent cations probed by site-directed mutation, optical spectroscopy, and kinetics
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DOI：
10.1021/bi002690p
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发表时间：
2001-03-27
期刊：
影响因子：
2.9
通讯作者：
Dunn, MF
中科院分区：
文献类型：
--
作者：
Weber-Ban, E;Hur, O;Dunn, MF
色氨酸合成酶双酶复合物是最广泛记录的底物通道的例子，其中低聚单元已被描述在近原子分辨率。共同代谢物吲哚在α-和β-位点之间的转移通过沿着25埃长的互连通道在α（2）β（2）寡聚体的每个α β-二聚体单元内扩散而发生.代谢物转移的控制涉及变构相互作用，其触发α β-二聚体单元在开放和闭合构象之间以及在低活性和高活性的催化状态之间的转换。这种变构信号是由β位点的共价转化和配体与α位点的结合引发的。信号通过结构元件的支架在位点之间传递，所述结构元件包括单价阳离子（MVC）结合位点和β Lys 167与β Asp 305或α Asp 56的盐桥接相互作用。通过这些氨基酸残基的定点突变和MVC位点的阳离子取代的组合策略，本工作检查MVC位点和形成在Lys β 167与Asp β 305或Asp α 56之间的替代盐桥对通道调节的相互关系。这些实验表明，MVC的结合和Lys β 167-Asp α 56盐桥的形成对于位点之间的变构信号的传递是重要的，而β K167和β D305之间的盐桥似乎对催化和变构调节仅具有次要意义。这些发现的机制的影响，无论是基板沟道和催化进行了讨论。
The tryptophan synthase bienzyme complex is the most extensively documented example of substrate channeling in which the oligomeric unit has been described at near atomic resolution. Transfer of the common metabolite, indole, between the alpha- and the beta -sites occurs by diffusion along a 25-Angstrom -long interconnecting tunnel within each alpha beta -dimeric unit of the alpha (2)beta (2) oligomer. The control of metabolite transfer involves allosteric interactions that trigger the switching of alpha beta -dimeric units between open and closed conformations and between catalytic states of low and high activity. This allosteric signaling is triggered by covalent transformations at the beta -site and ligand binding to the alpha -site. The signals are transmitted between sites via a scaffolding of structural elements that includes a monovalent cation (MVC) binding site and salt bridging interactions of beta Lys 167 with beta Asp 305 or alpha Asp 56. Through the combined strategies of site-directed mutations of these amino acid residues and cation substitutions at the MVC site, this work examines the interrelationship of the MVC site and the alternative salt bridges formed between Lys beta 167 with Asp beta 305 or Asp alpha 56 to the regulation of channeling. These experiments show that both the binding of a MVC and the formation of the Lys beta 167-Asp alpha 56 salt bridge are important to the transmission of allosteric signals between the sites, whereas, the salt bridge between beta K167 and beta D305 appears to be only of minor significance to catalysis and allosteric regulation. The mechanistic implications of these findings both for substrate channeling and for catalysis are discussed.