## 2 Regulation of CTPS Activity by GTP
### Kinetics Studies: Laying the Groundwork
Overall, these and other kinetics studies on the activation and inhibition of CTPS by GTP have been interpreted in terms of kinetic mechanisms similar to the one shown in Scheme 1, which yields the initial velocity Equation (1), where \(k_{\rm o}\) and \(k_{\rm act}\) are the rate constants for the formation of CTP in the absence and presence of GTP, respectively, \(K_{\rm A}\) is the dissociation constant for the E-GTP complex, \(K_{\rm i}{}^{n}\) is the apparent dissociation constant for the non-productive E-(GTP)\({}_{n}\) complex, and \(n\) is the Hill number [71; 72]. \[\frac{v_{\rm i}}{[\rm E]_{\rm T}}=\frac{\left(k_{\rm o}+\left(\frac{k_{\rm act }[\rm GTP]}{K_{\rm A}}\right)\right)[\rm Gln]}{\left(1+\frac{[\rm GTP]}{K_{ \rm A}}+\left(\frac{[\rm GTP]}{K_{\rm i}}\right)^{n}\right)\left(K_{\rm S}+[ \rm Gln]\right)} \tag{1}\]  
Typically, saturating concentrations of Gln are employed so that Equation (1) reduces to Equation (2) to simplify the kinetic analysis. \[\frac{v_{\rm i}}{[\rm E]_{\rm T}}=\frac{k_{\rm o}+\left(\frac{k_{\rm act}[\rm GTP ]}{K_{\rm A}}\right)}{1+\frac{[\rm GTP]}{K_{\rm A}}+\left(\frac{[\rm GTP]}{K _{\rm i}}\right)^{n}} \tag{2}\]  
Variations of Equation (1) have also been utilized such as Equation (3) [36; 73; 76]. \[\frac{v_{\rm i}}{[\rm E]_{\rm T}}=k_{\rm cat,1}+\frac{k_{\rm cat,2}[\rm GTP]}{K _{\rm A}+[\rm GTP]} \tag{3}\]  
The observation that GTP could inhibit the utilization of exogenous NH\({}_{3}\) in a concentration-dependent manner suggested that the binding of GTP could block the access of NH\({}_{3}\) to the enzyme and that the entry point for the exogenous NH\({}_{3}\) was located in the GAT domain. While GTP weakly inhibits NH\({}_{3}\)-dependent CTP formation by _Ec_CTPS at all concentrations [71], the inhibition is more pronounced when the active-site Cys is covalently modified by 6-diazo-5-oxo-l-norleucine (DON, Figure 1) [64], which mimics the glutamylenzyme intermediate, or when the enzyme is inhibited by glutamate \(\gamma\)-semialdehyde, which mimics the tetrahedral intermediate(s) formed during hydrolysis [75]. It is possible that the negative cooperativity reported by [PERSON] and [PERSON] [64] reflected the conformationalchanges that accompany the inhibition of both the NH\({}_{3}\)- and Gln-dependent CTP formation at elevated concentrations of GTP (vide infra) [71; 72]. Similar to _Ec_CTPS, GTP binding inhibited NH\({}_{3}\)-dependent CTP formation by DON-inactivated _L_CTPS, although, in this case, the inhibition by GTP exhibited negative cooperativity (\(K_{\text{i}}=0.40\) mM, \(n=0.39\)) [36]. Furthermore, GTP did not inhibit NH\({}_{3}\)-dependent CTP formation catalyzed by _L_ICTPS. In addition to these differences, _Ec_CTPS and _L_ICTPS exhibited differences in how Gln hydrolysis was regulated by GTP. While [PERSON] and [PERSON] [64] found that _Ec_CTPS catalyzed the GTP-activated hydrolysis of Gln in the presence of UTP and ADPNP at a steady-state rate similar to the rate of the GTP-activated synthesis of CTP and with a similar degree of enhancement in the presence of GTP, [PERSON] and [PERSON] [36] found that GTP-dependent activation of the _L_ICTPS-catalyzed glutaminase reaction never attained the same levels as the overall reaction for GTP-activated Gln-dependent CTP formation, including in the presence of UTP and the non-hydrolyzable ATP analogue adenosine-5\({}^{\prime}\)-[\(\gamma\)-thio] triphosphate (ATP\(\gamma\)S). Unlike _Ec_CTPS, the affinity of _L_ICTPS for GTP was increased in the presence of ATP and UTP. Indeed, the degree of saturation with ATP and UTP, unlike ATP\(\gamma\)S and UTP, had a pronounced influence on the \(K_{\text{A}}\) value for GTP binding, with the value of \(K_{\text{A}}\) decreasing with lower concentrations of the substrate nucleotides [73]. Moreover, at low GTP concentrations ranging from \(\sim\)10 to 100 \(\upmu\)M, the rate of Gln hydrolysis exceeded that of CTP synthesis, resulting in uncoupling of the two reactions.