## 2 Regulation of CTPS Activity by GTP  
### Kinetics Studies: Laying the Groundwork  
CTPS exhibits an exquisite ability to distinguish between all four ribonucleotide-5\({}^{\prime}\)-triphosphates in varying roles, including substrates, activators, inhibitors, and effectors of oligomerization and filament formation. Consequently, CTPS has served as a paradigm for understanding the complexities of protein regulation. Although it has been roughly 50 years since it was established that GTP serves as a positive allosteric effector of CTPS [64; 65], enhancing the rate of Gln hydrolysis both in the presence and absence of substrate nucleotides, only within the last 15 years has structural information become available that provides atomic-level insights into the mechanism of allosteric activation. The first X-ray crystal structures of _Ec_CTPS with bound CTP and ADP [32; 44] and _Thermus thermophilus_ HB8 CTPS (_T_CTPS) with bound Gln [45] were reported in the mid-2000s, followed by the structures of apo-CTPS from _Sulfolous solitaticus_ (_Ss_CTPS) [67], the synthase domain of human CTPS [68], _Ec_CTPS with CTP and Gln bound [56], _Mycobacterium tuberculosis_ CTPS (_Mi_CTPS) with two UTP molecules bound or UTP, AMP-PCP, and 6-diaz-5-oxo-\(\iota\)-norleucine (DON) bound [15], and the glutaminase domain of _Tryanosoma brucei_ CTPS (_T_bCTPS) with bound acivicin [8]. Subsequently, cryo-electron microscopy (EM) structures were reported for filaments of human CTPS [56; 61] and _Drosophila melanogaster_ CTPS (_Dm_CTPS) [69]. However, none of these structures contained bound GTP and, hence, insights into the details of GTP-dependent activation at the atomistic level remained elusive until very recently [66]. In the absence of structural information delineating the exact conformational changes that GTP binding effects to stimulate the glutaminase activity, site-directed mutagenesis and kinetics studies were crucial in defining the location and functional effects of GTP binding. Early kinetics studies conducted by [PERSON] and [PERSON] revealed that GTP accelerates the glutaminase activity but has little or no effect on the rate of NH\({}_{3}\)-dependent CTP formation [64]. Although the original kinetic model for the effects of GTP on catalysis by _Ec_CTPS appeared to be quite complex, apparently involving both negative and positive cooperativity accompanying equilibrium binding experiments [64], later studies revealed that the activation of Gln-dependent CTP formation by GTP was hyperbolic for _Ec_CTPS [70; 71; 72], _Lactococcus lactis_ CTPS (_L_UCTPS) [36; 73], and the two yeast isozymes [74] under conditions where the enzyme was in its fully active, tetrameric state. That said, [PERSON] and [PERSON] concluded that GTP likely exerted its effect by enhancing the rate of formation of the glutamyl-enzyme intermediate from the E-Gln Michaelis complex with little effect on the \(K_{\rm m}\) value for Gln. Consistent with this hypothesis, [PERSON] and co-workers showed that GTP binding enhanced the inhibition of _Ec_CTPS by the intermediate/transition state analogue inhibitor glutamate \(\gamma\)-semialdehyde, thereby suggesting that GTP promotes the glutaminase activity by stabilizing a protein conformation that binds the tetrahedral intermediate(s) formed during Gln hydrolysis [75]. Similar results were obtained by [PERSON] with _L_UCTPS [76]. The notion that the allosteric binding of GTP causes subtle conformation changes and re-organization of the environment at the oxyanion hole to facilitate the glutaminase reaction was consistent with the behavior of other amidotransferases [77; 78; 79]. Later studies revealed that at elevated concentrations, GTP behaved as a weak negative allosteric effector of _Ec_CTPS inhibiting Gln-dependent CTP formation [71]. Interestingly, GTP did not inhibit the enzyme's intrinsic glutaminase activity. Furthermore, GTP inhibited NH\({}_{3}\)-dependent CTP formation.