折叠无关的界面残基对于I类谷氨酰胺酰胺转移酶中的复合物形成和变构信号至关重要。,Biochemistry

当前位置： X-MOL 学术 › Biochemistry › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

折叠无关的界面残基对于I类谷氨酰胺酰胺转移酶中的复合物形成和变构信号至关重要。
Biochemistry ( IF 2.9 ) Pub Date : 2019-05-23 , DOI: 10.1021/acs.biochem.9b00286
Florian Semmelmann ₁ , Enrico Hupfeld ₁ , Leonhard Heizinger ₁ , Rainer Merkl ₁ , Reinhard Sterner ₁

Affiliation

谷氨酰胺酰胺转移酶（GATase）家族的成员催化氨在许多代谢途径中的掺入，可以分为两类。在这里，我们集中于I类GATases，它们是由合成酶亚基和谷氨酰胺酶亚基与催化的Cys-His-Glu三联体组成的异聚酶复合物。谷氨酰胺酶亚基的谷氨酰胺水解取决于（i）依赖于紧密合酶-谷氨酰胺酶复合物的形成，和（ii）与合酶亚基的底物存在变构偶联。复杂的形成和变构的结构基础了解甚少。但是，先前关于4-氨基-4-脱氧胆酸合酶和咪唑甘油磷酸合酶的研究表明，它们的合酶亚基中存在保守的天冬氨酸残基，它位于靠近谷氨酰胺酶催化三联体的亚基界面处，对这两个功能都可能很重要。我们从蛋白质数据库中进行了I类GATases的计算筛选，并确定了保守且位置相似的天冬氨酸残基。然后，我们生成了这些残基的丙氨酸和谷氨酸突变体，并通过分析性凝胶过滤和稳态酶动力学对其进行了表征。结果证实了野生型天冬氨酸残基对于形成稳定的合酶-谷氨酰胺酶复合物（四分之三）和刺激谷氨酰胺酶活性（在所有四例中）具有重要作用。我们提出了一个模型，用于使保守的天冬氨酸残基对整个I类GATase家族的统一调节机制具有双重作用。

"点击查看英文标题和摘要"

A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit. The structural basis of both complex formation and allostery is poorly understood. However, previous work on 4-amino-4-deoxychorismate synthase and imidazole glycerol phosphate synthase suggested that a conserved aspartate residue in their synthase subunits, which is located at the subunit interface close to the glutaminase catalytic triad, might be important for both features. We performed a computational screen of class I GATases from the Protein Data Bank and identified conserved and similarly located aspartate residues. We then generated alanine and glutamate mutants of these residues and characterized them by analytical gel filtration and steady-state enzyme kinetics. The results confirmed the important role of the wild-type aspartate residues for the formation of stable synthase-glutaminase complexes (in three of four cases) and the stimulation of glutaminase activity in the analyzed GATases (in all four cases). We present a model for rationalizing the dual role of the conserved aspartate residue toward a unifying regulation mechanism in the entire class I GATase family.

更新日期：2019-05-22

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南