## 2 Regulation of CTPS Activity by GTP
### Regulatory Effects of NADH  
[PERSON], [PERSON], [PERSON], and co-workers observed that nicotinamide-containing compounds were modest inhibitors of _Ec_CTPS with IC\({}_{50}\) values following the trend: 1-methyl 1,4-dihydronicotinamide (IC\({}_{50}\) = 140 uM) \(<\) NADH \(\approx\) NADPH \(<<\) NADP\({}^{+}\) \(<\) NAD\({}^{+}\)\(<<\) 1-methyl nicotinamide (IC\({}_{50}\)-4000 uM), indicating that the reduced nicotinamide ring is sufficient for inhibition [87]. NADH inhibition was enhanced in the presence of increasing concentrations of GTP, including those concentrations for which GTP is either activating or inhibitory. Correspondingly, in the presence of a fixed concentration of NADH, the dependence of the activating and inhibitory effects of GTP were more acute (i.e., lowering both the EC\({}_{50}\) and IC\({}_{50}\) values for activation and inhibition, respectively). Hence, NADH enhanced the allosteric regulation of _Ec_CTPS by GTP in a mutual fashion. Furthermore, these authors found that increasing the concentration of GTP enhanced the inhibition by CTP; conversely, in the presence of CTP, the EC\({}_{50}\) and IC\({}_{50}\) values for GTP were reduced. The observations that NADH, CTP, and GTP mutually enhanced each other's effects suggested that they interact with a common intermediate enzyme state. Finally, [PERSON] et al. [53] demonstrated that CTP was a more effective inhibitor under conditions that favored filament formation by the enzyme (i.e., elevated concentrations of CTP or the enzyme). Using the E227R _Ec_CTPS variant, which obviates the ability of the enzyme to form filaments and reduces the inhibitory effects of CTP, [PERSON], [PERSON], [PERSON], and co-workers observed that the EC\({}_{50}\) and IC\({}_{50}\) values were shifted to higher values for GTP-dependent activation and inhibition, respectively [87]. This intriguing observation suggested that GTP interacted with the inhibitory filament, which was an insightful claim, considering that at that point, GTP had not been observed in any of the high-resolutioncryo-EM reconstructions of CTPS filaments [87]. Although NADH did not appear to bind at the UTP- and CTP-binding sites, it remains unclear where NADH binds to exert its effects on the GTP-dependent activity of _Ec_CTPS [44; 87]. Overall, these observations highlight the important regulatory role that GTP plays by inducing conformational changes that affect the binding and activity of other nucleotide ligands, which is also consistent with the notion that the 4-phospho-UTP and GTP could act as coactivators of the glutaminase reaction as pointed out by [PERSON] [36]. Structural Studies Intimating the Location of the GTP-Binding Site and the Role of GTP-Dependent Activation in NH\({}_{3}\) Translocation  
A breakthrough in the study of CTPS occurred when the first X-ray crystal structures of CTPS were reported in 2004. [PERSON] and co-workers reported the structure of _Ec_CTPS in its ligand-free form [32] and [PERSON] and co-workers reported the structure of _T_CTPS with bound Gln [45]. A year later, [PERSON]'s laboratory reported the structure of the _Ec_CTPS-ADP-CTP product complex [44]. These seminal structures helped to delineate the spatial arrangement of the site of Gln hydrolysis, the NH\({}_{3}\) tunnel, the binding of nucleotides at the synthase site, and the protein oligomerization interfaces, as well as inform the design and interpretation of site-directed mutagenesis studies. Unfortunately, no electron density for GTP was present, despite GTP being present in the crystallization solution for the _Ec_CTPS structures [44; 87]. Hence, few direct insights were provided regarding the conformational changes accompanying GTP binding. However, by comparing the GAT domain with structurally related GTP-binding proteins (e.g., EF-Tu and EF-G), [PERSON] and co-workers [32] identified a potential binding site for GTP located adjacent to the entrance to the glutaminase active site in a deep cleft residing between the GAT and synthase domains close to the A-A' dimer interface (Figure 3).