Thoden J B, Miran S G, Phillips J C, Howard A J, Raushel F M, Holden H M
Institute for Enzyme Research, The Graduate School, University of Wisconsin-Madison 53705, USA.
Biochemistry. 1998 Jun 23;37(25):8825-31. doi: 10.1021/bi9807761.
Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine. The enzyme consists of two polypeptide chains referred to as the large and small subunits. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes. Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis. Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E. coli in which His 353 has been replaced with an asparagine residue. Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution. The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.
来自大肠杆菌的氨甲酰磷酸合成酶催化由两分子Mg2 + ATP、一分子碳酸氢盐和一分子谷氨酰胺生成氨甲酰磷酸。该酶由两条多肽链组成，分别称为大亚基和小亚基。大亚基提供负责结合核苷酸和其他效应配体的活性位点，而小亚基包含催化谷氨酰胺水解为谷氨酸和氨的氨基酸残基。从氨基酸序列分析和结构研究可知，小亚基属于I类酰胺转移酶家族。大量生化研究表明，小亚基的反应机制通过谷氨酰硫酯中间体的形成进行，并且Cys 269和His 353对催化作用都至关重要。在此，我们描述了大肠杆菌氨甲酰磷酸合成酶的X射线晶体结构，其中His 353已被天冬酰胺残基取代。研究中使用的晶体是在谷氨酰胺存在下生长的，并且该模型已针对从30至1.8 Å分辨率的所有测量X射线数据精修至晶体学R因子为19.1%。小亚基的活性位点在Cys 269处明显含有一个共价结合的硫酯中间体，实际上，这项研究首次直接观察到了酰胺转移酶家族中酶中间体的结构。
