Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada.
Biomolecules. 2022 Apr 29;12(5):647. doi: 10.3390/biom12050647.
Cytidine-5'-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible "wing" region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
三磷酸胞苷合酶（CTPS）是 I 类依赖于谷氨酰胺的氨基转移酶（GAT），可催化从头合成三磷酸胞苷的最后一步。谷氨酰胺水解在 GAT 结构域中进行，释放的氨通过分子内隧道转移到合成酶结构域，在那里 UTP 依赖于 ATP 的氨化形成 CTP。CTPS 在依赖于谷氨酰胺的氨基转移酶中是独特的，需要别构效应物（GTP）来激活 GAT 结构域，以有效地进行谷氨酰胺水解。最近，首次解析了结合了 ATP、UTP 以及显著的 GTP 的 CTPS 的冷冻电子显微镜结构，以及与 6-重氮-5-氧代-L-正亮氨酸的共价加合物。这些结构信息，以及过去 50 年来进行的大量定点突变、动力学和结构研究，提供了更详细的信息，了解 GTP 结合到 GAT 结构域时伴随的精细构象变化，以及它们对催化的贡献。GTP 与 L2 环、来自相邻亚基的 L4 环、L11 盖和 L13 环（或独特的灵活“翼”区域）之间的相互作用诱导构象变化，促进 GAT 结构域中谷氨酰胺的水解；然而，关于这些构象变化如何促进 GAT 结构域催化的确切机制的直接实验证据仍然缺乏。重要的是，GTP 结合诱导的构象变化也会影响 NH 隧道的组装和维持。因此，除了促进谷氨酰胺水解外，别构效应物在协调 CTPS 的 GAT 和合成酶结构域催化的反应中起着重要作用。
