Thoden J B, Wesenberg G, Raushel F M, Holden H M
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53705, USA.
Biochemistry. 1999 Feb 23;38(8):2347-57. doi: 10.1021/bi982517h.
Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.
氨甲酰磷酸合成酶（CPS）催化氨甲酰磷酸的生成，随后氨甲酰磷酸用于嘧啶核苷酸或精氨酸合成的代谢途径。该酶的催化机制通过三种高活性中间体发生：羧基磷酸盐、氨和氨基甲酸盐。从大肠杆菌中分离出来的CPS是一种α、β异二聚体蛋白，其三个活性位点相距近100埃。此外，还有别构调节剂、鸟氨酸和UMP的独立结合位点。鉴于三个活性位点和别构结合口袋之间的距离较大，有人推测结构域运动在分子内通讯中起关键作用。在此，我们描述了来自大肠杆菌的CPS的结构，实际上，这种结构域运动是响应核苷酸结合而发生的。具体而言，该蛋白在不可水解类似物AMPPNP存在下结晶，并通过X射线晶体学分析确定其结构分辨率为2.1埃。大亚基的氨甲酰磷酸合成成分的B结构域在活性位点口袋上方关闭，使得一些原子相对于原始结构中观察到的移动超过7埃。这种运动的触发因素在于两个主链酰胺基团（Gly 721和Gly 722）与三磷酸核苷酸的β和γ磷酸基团之间的氢键相互作用。Gly 721和Gly 722位于III'型反向转角中，并且在D-丙氨酸：D-丙氨酸连接酶和谷胱甘肽合成酶中也观察到这种二级结构基序，这两种酶都属于“ATP抓握”蛋白超家族。描述了CPS大亚基中包含的两个活性位点的几何细节。
