Long-range allosteric transitions in carbamoyl phosphate synthetase.
Long-range allosteric transitions in carbamoyl phosphate synthetase.
复制标题
氨基甲酰磷酸合成酶中的长程变构转变。
DOI：
10.1110/ps.04822704
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Holden,HazelM
中科院分区：
文献类型：
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作者：
Thoden,JamesB;Huang,Xinyi;Kim,Jungwook;Raushel,FrankM;Holden,HazelM
氨甲酰磷酸合成酶通过催化一分子碳酸氢盐、两分子MgATP和一分子谷氨酰胺产生氨甲酰磷酸，在嘧啶和精氨酸生物合成中起关键作用。大肠杆菌中的酶由两条多肽链组成，称为小亚基和大亚基，它们总共含有三个独立的活性位点，这些位点通过分子内通道连接。小亚基含有这些活性位点之一，负责将谷氨酰胺水解为谷氨酸和氨。大亚基结合MgATP所需的两个分子，并参与组装最终产物。化合物如L-鸟氨酸、UMP和IMP变构调节酶。在这里，我们报告的三维结构的定点突变蛋白的氨甲酰磷酸合成酶弗罗姆。coli中，其中小亚基中的Cys 248被改变为天冬氨酸。将该残基作为结构研究的目标，因为先前的研究表明，C248 D突变蛋白的部分丙氨酸氨基转移酶活性相对于野生型酶增加了40倍，而使用谷氨酰胺作为氮源形成的氨基甲酰磷酸被完全消除。值得注意的是，虽然小亚基中的Cys 248位于大亚基中的变构结合口袋的100 nm处，但电子密度图清楚地显示了UMP的存在，尽管该配体从未包括在纯化或结晶方案中。描述了UMP与氨甲酰磷酸合成酶结合的方式。
Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme fromEscherichia coliconsists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L‐ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three‐dimensional structure of a site‐directed mutant protein of carbamoyl phosphate synthetase fromE. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40‐fold relative to the wild‐type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at ∼100 Å from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described.