Byrnes W M, Hu W, Younathan E S, Chang S H
Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
J Biol Chem. 1995 Feb 24;270(8):3828-35. doi: 10.1074/jbc.270.8.3828.
The phosphofructokinases (PFKs) from the bacteria Escherichia coli and Bacillus stearothermophilus differ markedly in their regulation by ATP. Whereas E. coli PFK (EcPFK) is profoundly inhibited by ATP, B. stearothermophilus PFK (BsPFK) is only slightly inhibited. The structural basis for this difference could be closure of the active site via a conformational transition that occurs in the ATP-binding domain of EcPFK, but is absent in BsPFK. To investigate the role of this transition in ATP inhibition of EcPFK, we have constructed a chimeric enzyme that contains the "rigid" ATP-binding domain of BsPFK grafted onto the remainder of the EcPFK subunit. The chimeric PFK has the following characteristics: (i) tetrameric structure and kinetic parameters similar to those of the native enzymes, (ii) insensitivity to regulation by the effector phosphoenolpyruvate despite its ability to bind to the enzyme, and (iii) a sigmoidal (nH around 2) fructose 6-phosphate saturation curve. From the results, it is concluded that the active site regions of the two native enzymes are remarkably similar, but their effector sites and their mechanisms of heterotropic regulation are different. The chimeric subunit is locked in a structure resembling that of activated E. coli PFK, yet the enzyme can exist in two different conformational states. Mechanisms for its sigmoidal kinetics are discussed.
来自大肠杆菌和嗜热栖热芽孢杆菌的磷酸果糖激酶（PFK）在受ATP调节方面存在显著差异。大肠杆菌PFK（EcPFK）受到ATP的强烈抑制，而嗜热栖热芽孢杆菌PFK（BsPFK）仅受到轻微抑制。这种差异的结构基础可能是EcPFK的ATP结合结构域中发生的构象转变导致活性位点关闭，而BsPFK中不存在这种转变。为了研究这种转变在ATP对EcPFK抑制中的作用，我们构建了一种嵌合酶，它含有嫁接到EcPFK亚基其余部分上的BsPFK的“刚性”ATP结合结构域。这种嵌合PFK具有以下特征：（i）四聚体结构和动力学参数与天然酶相似；（ii）尽管能够与效应物磷酸烯醇丙酮酸结合，但对其调节不敏感；（iii）具有S形（nH约为2）的6-磷酸果糖饱和曲线。从结果可以得出结论，两种天然酶的活性位点区域非常相似，但它们的效应物位点和异源调节机制不同。嵌合亚基被锁定在类似于活化的大肠杆菌PFK的结构中，但该酶可以以两种不同的构象状态存在。文中讨论了其S形动力学的机制。
