###### Abstract  
Cytidine-5\({}^{\prime}\)-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 amitation 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 _Drosophila_ 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\({}_{3}\) 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. C 20  
(_Ec_CTPS) residues 287-544) [31; 32]), which is then transferred through an intramolecular tunnel to the N-terminal synthase (or amidoligase) domain (_Ec_CTPS residues 1-266 connected to the GAT domain via an interdomain linker, residues 267-286) [32]. The lack of equilibration of the nascent NH\({}_{3}\) with the solvent supported the notion of such a tunnel [33]. Upon entering the synthase domain, the nascent NH\({}_{3}\) reacts at the 4-position of 4-phospho-UTP, formed through the ATP-dependent phosphorylation of UTP [34; 35; 36], to yield CTP (Figure 1) [37]. The enzyme can also utilize exogenous NH\({}_{3}\) directly as a nitrogen source [38]. Considering the central role of CTP in metabolism, it is not surprising that CTPS is highly regulated. While the substrates ATP and UTP promote oligomerization of the enzyme from inactive monomers and dimers to active tetramers [37; 39; 40; 41; 42; 43] by binding at an interfacial active site formed by the synthase domains [32; 44; 45], the product CTP can also induce tetramerization [43] as well as act as a feedback inhibitor [37; 44]. Eukaryotic homologues of CTPS are also regulated by phosphorylation [46; 47; 48; 49; 50; 51]. More recently, CTPS has been shown to form a superhelix of tetramers, and this filamentous structure (also known as cytopohidium) constitutes an additional level of regulation [52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62]. Finally, guanosine-5\({}^{\prime}\)-triphosphate (GTP) is an allosteric regulator of CTPS activity [37; 63; 64; 65]. Indeed, among the Gln-dependent amidotransferases, CTPS is unique in that it is the only member of this family of enzymes wherein the GAT domain requires an allosteric effector for the efficient hydrolysis of Gln. Initially, GTP was believed to simply promote the Gln hydrolysis reaction; however, over the past two decades, kinetics, site-directed mutagenesis, and structural studies have revealed that the role of GTP is much more complex than originally thought.