Disentangling the web of allosteric communication in a homotetramer:: Heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus
Disentangling the web of allosteric communication in a homotetramer:: Heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus
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DOI：
10.1021/bi035077p
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发表时间：
2004-01-20
期刊：
影响因子：
2.9
通讯作者：
Reinhart, GD
中科院分区：
文献类型：
--
作者：
Ortigosa, AD;Kimmel, JL;Reinhart, GD
本文描述了一种分离嗜热脂肪芽孢杆菌同四聚体磷酸果糖激酶中存在的四种可能独特的异异性对变构相互作用的策略。该策略包括构建包含一个野生型亚基和三个突变亚基的杂交四聚体，这些亚基被修饰以阻断底物果糖6-磷酸（Fru-6-P）和变构抑制剂磷酸（烯醇）丙酮酸（PEP）的结合。每种类型的结合位点都发生在亚基界面上，并且已经确定了界面两侧的突变，这些突变将大大减少各自位点的结合。因此，已经创建了四种不同类型的突变亚基，每种亚基都含有不同的活性位点和变构位点修饰。相应的1:3杂交体分离出一对不同的未修饰底物和具有独特结构配置的变构位点，分别位于22、30、32和45埃之间。在这四种杂交体中，未修饰位点所表现出的变构抑制作用已经用耦合自由能进行了定量评价。每个偶联自由能的大小都是唯一的，它们的相对大小随pH值的变化而变化。重要的是，每个pH下这些偶联自由能的总和等于与四聚体酶相关的总异向偶联自由能。后者的数量是通过去除PEP结合中的同向相互作用的对照杂交种的总体抑制来评估的。结果不同意协调或顺序模型，往往被用来解释在低聚酶的变构行为。
A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 Angstrom apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.