Thoden James B, Huang Xinyi, Raushel Frank M, Holden Hazel M
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, 53706-1544, USA.
J Biol Chem. 2002 Oct 18;277(42):39722-7. doi: 10.1074/jbc.M206915200. Epub 2002 Jul 18.
Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of approximately 100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.
氨甲酰磷酸合成酶通过一种反应机制催化氨甲酰磷酸的生成，该反应机制需要一分子碳酸氢盐、两分子MgATP和一分子谷氨酰胺。来自大肠杆菌的这种酶由两条多肽链组成。其中较小的一条属于I类酰胺转移酶超家族，包含将谷氨酰胺水解为谷氨酸和氨所需的所有必要氨基酸侧链。较大亚基的两个同源结构域采用了ATP结合超家族成员特有的构象。这些ATP结合结构域中的每一个都含有一个负责结合一分子MgATP的活性位点。高分辨率X射线晶体学分析表明，值得注意的是，大肠杆菌酶中的三个活性位点通过一条总长约100埃的分子隧道相连。在这里，我们描述了氨甲酰磷酸合成酶G359F（小亚基）突变蛋白的高分辨率X射线晶体结构。最初选择这个残基进行研究是因为它位于从小亚基活性位点通向大亚基第一个活性位点的分子隧道内壁。预计将其突变为较大的残基会“堵塞”氨隧道，并阻碍氨从其产生位点向利用位点的传递。事实上，G359F取代导致了由Glu-355至Ala-364界定的环的构象发生完全变化，从而为氨中间体直接通向大量溶剂提供了一条“逃逸”途径。该取代还影响了小亚基活性位点中几个关键催化氨基酸侧链的排布。
