Ortigosa Allison D, Kimmel Jennifer L, Reinhart Gregory D
Department of Biochemistry and Biophysics and Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA.
Biochemistry. 2004 Jan 20;43(2):577-86. doi: 10.1021/bi035077p.
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 A 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.
本文描述了一种策略，用于分离嗜热脂肪芽孢杆菌同源四聚体磷酸果糖激酶中存在的四种潜在独特的异源成对变构相互作用。该策略涉及构建包含一个野生型亚基和三个突变亚基的杂合四聚体，这些突变亚基经过修饰以阻断底物6-磷酸果糖（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出现在亚基界面处，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，创建了四种不同类型的突变亚基，每个亚基都包含不同的活性位点和变构位点修饰。相应的1:3杂合体分别分离出一对不同的未修饰底物和变构位点，它们具有独特的结构布局，彼此相距22、30、32和45埃。这四种杂合体中每个未修饰位点所表现出的变构抑制作用已根据耦合自由能进行了定量评估。每个耦合自由能的大小都是独特的，并且它们的相对大小随pH值变化。重要的是，在每个pH值下，这些耦合自由能的总和等于与四聚体酶相关的总异源耦合自由能。后者的量是通过去除PEP结合中的同促相互作用的对照杂合体的总体抑制作用来评估的。结果与通常用于解释寡聚酶变构行为的协同模型或顺序模型均不一致。
