Thoden James B, Huang Xinyi, Kim Jungwook, Raushel Frank M, Holden Hazel M
Department of Biochemistry, University of Wisconsin, Madison, WI, 53706, USA.
Protein Sci. 2004 Sep;13(9):2398-405. doi: 10.1110/ps.04822704.
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 from Escherichia coli consists 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 from E. 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 approximately 100 A 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.
氨甲酰磷酸合成酶在嘧啶和精氨酸生物合成中都起着关键作用，它催化一分子碳酸氢盐、两分子MgATP和一分子谷氨酰胺生成氨甲酰磷酸。大肠杆菌中的这种酶由两条多肽链组成，分别称为小亚基和大亚基，它们总共包含三个独立的活性位点，这些位点通过分子内隧道相连。小亚基包含其中一个活性位点，负责将谷氨酰胺水解为谷氨酸和氨。大亚基结合两个所需的MgATP分子，并参与最终产物的组装。L-鸟氨酸、UMP和IMP等化合物对该酶具有别构调节作用。在此，我们报道了大肠杆菌氨甲酰磷酸合成酶定点突变蛋白的三维结构，其中小亚基中的半胱氨酸248被替换为天冬氨酸。选择该残基进行结构研究是因为先前的研究表明，C248D突变蛋白的部分谷氨酰胺酶活性相对于野生型酶提高了40倍，而以谷氨酰胺为氮源生成氨甲酰磷酸的过程则完全被阻断。值得注意的是，尽管小亚基中的半胱氨酸248距离大亚基中的别构结合口袋约100 Å，但电子密度图清楚地显示了UMP的存在，尽管该配体从未包含在纯化或结晶方案中。本文描述了UMP与氨甲酰磷酸合成酶结合的方式。
