Liu X, Guy H I, Evans D R
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 1994 Nov 4;269(44):27747-55.
Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.
来自不同生物体的氨甲酰磷酸合成酶具有相似的催化机制和氨基酸序列，但其结构组织、亚基结构和调节方式可能有很大差异。大肠杆菌氨甲酰磷酸合成酶（CPSase）是一种由酰胺转移酶和合成酶亚基组成的单功能蛋白质，受UMP变构抑制，受NH3、IMP和鸟氨酸激活。相比之下，哺乳动物CPSase II是大型多功能多肽CAD的一部分，受UTP抑制，受5-磷酸核糖-1-焦磷酸（PRPP）激活。先前对大肠杆菌CPSase的光亲和标记研究表明，变构效应物结合在合成酶亚基的羧基末端附近。分子的这一区域可能是所有CPSase共有的调节亚结构域。构建了一个大肠杆菌-哺乳动物杂交CPSase基因并在大肠杆菌中表达。该杂交体由大肠杆菌CPSase合成酶催化亚结构域（1073个氨基酸多肽中的第1至900位残基）与哺乳动物蛋白质假定的190个氨基酸调节亚结构域的氨基末端融合而成。杂交CPSase具有正常活性，但不再受原核变构效应物的调节。相反，谷氨酰胺和氨依赖性CPSase活性以及两个ATP依赖性部分反应均被PRPP激活并被UTP抑制，这表明这两种配体的结合位点位于CAD的CPSase结构域羧基末端的调节区域。杂交体和野生型哺乳动物蛋白质中UTP的表观配体解离常数和抑制程度相似，但PRPP与杂交体的结合亲和力弱4倍。变构配体对杂交体的稳态动力学参数影响不同，这表明虽然催化亚结构域和调节亚结构域之间的联系得以保留，但结构域间信号传递可能存在质的差异。然而，原核和真核变构控制的切换表明该蛋白质家族在结构和调节机制上具有显著的保守性。
