The allosteric regulation of pyruvate kinase - A site-directed mutagenesis study
The allosteric regulation of pyruvate kinase - A site-directed mutagenesis study
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DOI：
10.1074/jbc.m001870200
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发表时间：
2000-06-16
影响因子：
4.8
通讯作者：
Mattevi, A
中科院分区：
文献类型：
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作者：
Valentin, G;Chiarelli, L;Mattevi, A
丙酮酸激酶（PK）是糖酵解途径的关键调节因子。大肠杆菌的调节特性仅通过突变涉及结构域间盐桥（Arg（271）、Arg（292）、Asp（297）和Lys（413））和结合变构激活剂（Lys（382）和Arg（431））的6个带电残基来研究。Arg（271）和Lys（413）位于一个亚基内A和C结构域之间的界面处。R271 L和K413 Q突变体酶表现出改变的动力学性质。在K413 Q中，存在部分酶激活，而R271 L的特征是在变构平衡中偏向T状态。在T状态下，Arg（292）和Asp（297）形成亚基间盐桥。突变体R292 D和D297 R完全失活。R292 D的晶体结构表明，突变酶保留了T-状态的四级结构。然而，突变诱导重组的接口与创建一个网络的相互作用类似的R-状态酵母和M1 PK蛋白的晶体结构中观察到的。此外，在R292 D结构中，作为活性位点的一部分的两个环是无序的。K382 Q和R431 E突变被设计用于探测果糖1，6-二磷酸（变构激活剂）的结合位点。R431 E在调节特性方面仅表现出轻微的变化。相反，K382 Q显示出对激活剂的高度改变的反应性，表明Lys（382）参与激活剂结合和变构转换机制。两者合计，这些结果支持的概念，域接口是至关重要的变构转换。它们将三级和四级结构的变化与果糖1，6-二磷酸和底物结合位点的几何结构的改变偶联。这些定点突变的数据进行了讨论的遗传性非球形红细胞溶血性贫血，这是由人红细胞PK基因突变的分子基础。
Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli mere investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit, The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1,6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1,6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.