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首页> 外文OA文献 >Functional role of fumarate site Glu59 involved in allosteric regulation and subunit-subunit interaction of human mitochondrial NAD(P)(+)-dependent malic enzyme
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Functional role of fumarate site Glu59 involved in allosteric regulation and subunit-subunit interaction of human mitochondrial NAD(P)(+)-dependent malic enzyme

机译：Functional role of fumarate site Glu59 involved in allosteric regulation and subunit-subunit interaction of human mitochondrial NaD(p)(+)-dependent malic enzyme

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摘要

Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, K-A,K-Fum and alpha K-A,K-Fum. The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The K-A,K-Fum and alpha K-A,K-Fum of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer-tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer-tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP(+)-dependent malic enzyme and cytosolic NADP(+)-dependent malic enzyme in fumarate activation and malate cooperativity.
机译：在这里，我们报告在人类线粒体NAD（P）（+）依赖性苹果酸酶（m-NAD-ME）的富马酸酯介导的变构调节中Glu59的作用。在本研究中，Glu59被Asp，Gln或Leu取代。我们的动力学数据强烈表明，该残基的电荷性质会显着影响酶的变构活化。 E59L酶显示出非变构动力学，E59Q酶显示出更高的活化常数阈值，具有较高的活化常数K-A，K-Fum和αK-A，K-Fum。 E59D酶尽管保留了变构性质，但在酶激活方面与野生型有很大不同。 E59D的KA，K-Fum和alpha KA，K-Fum也比野生型大得多，这表明不仅该残基的负电荷而且基团特异性和侧链相互作用对于富马酸酯也很重要捆绑。超离心分析表明，野生型和E59Q酶均以二聚体-四聚体平衡形式存在。与E59Q突变体相反，E59D突变体主要显示为二聚体形式，表明通过将Glu59的一条碳侧链缩短为Asp59可以改变二聚体界面的季稳定性。 E59L酶也显示出类似于野生型的二聚体-四聚体模型，但是它显示出更多的二聚体以及单体和聚合物。苹果酸的协同作用在E59突变酶中并不显着，这表明该协同作用可能与富马酸酯结合位点的分子几何结构有关。 Glu59可以精确地维持底物协同作用的几何特异性。根据序列比对分析和我们的实验数据，我们认为电荷效应和几何特异性都是酶调节的关键因素。 Glu59区分人类m-NAD-ME与线粒体NADP（+）依赖性苹果酸酶和胞质NADP（+）依赖性苹果酸酶富马酸酯的活化和苹果酸的协同作用。

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