## 4 GTP-Dependent Activation of CTPSs from Other Species
### Mammals  
Human CTPS exists as two isoforms, hCTPS1 and hCTPS2 [109; 110]. Comparison of the effects of GTP concentration on hCTPS1 and hCTPS2 activity revealed that GTP stimulated Gln-dependent CTP formation with \(K_{\rm A}\) values of 1.6 and 8.0 uM, respectively, while concentrations of GTP exceeding 10 mM inhibited the activity of both isoforms [100]. Earlier studies with hCTPS isolated from homogenates of HL-60 cells revealed negative cooperativity with GTP (\(n\) = 0.7). However, this was a crude preparation and likely contained a mixture of isoforms [111].  
Initial studies on CTPS purified 200-fold from Ehrlich ascites tumor cells (mouse) revealed that the enzyme was active in the absence of GTP and that GTP activated the enzyme \(\sim\)5-fold with maximal activation occurring at 1.0 mM GTP (\(K_{\rm A}\) = 50 uM) [104]. The \(K_{\rm m}\) value for Gln was not significantly altered by the presence of GTP. GTP had a slight inhibitory effect on the activity with exogenous NH\({}_{3}\). Follow-up studies showed that GTP accelerated the glutaminase activity of the enzyme about 2.5-fold in the absence of other substrates and about 45-fold in the presence of UTP and ATP [65]. Curiously, these authors also reported that GTP could substitute for ATP as a substrate (\(K_{\rm m}\) = 2.2 mM), even after confirming by HPLC analyses that their UTP and GTP preparations were both free of any ATP impurity. GMP, GDP, and dGTP were also capable of partially activating the enzyme, with the latter compound also serving to replace ATP [105]. Finally, the non-hydrolyzable GTP analogue Gpp[NH]p was shown to activate the enzyme by stimulating the glutaminase activity only \(\sim\)1.4-fold or \(\sim\)1.8-fold in the absence or presence, respectively, of both UTP and App[NH]p. However, in the presence of UTP and ATP, the glutaminase activity with concomitant CTP formation was enhanced \(\sim\)32-fold. These results are not unlike those obtained by [PERSON] and co-workers working with _LI_CTPS [36], indicating that the presence of UTP and ATP is required for the maximum activation of the glutaminase activity by GTP or Gpp[NH]p.  
Bovine calf liver CTPS was shown to not have an absolute requirement for GTP, but in the absence of GTP, the rate was reduced \(\sim\)7-fold relative to the rate observed at saturating concentrations of GTP [63; 101]. The \(K_{\rm A}\) value was reported to be 70 uM (\(n\) = 1.1), the same as the apparent \(K_{\rm A}\) value accompanying GTP-dependent activation of crude preparations of rat liver CTPS (Table 2) [18]. GTP did not promote tetramerization of the enzyme [63; 101; 102].  
Among the various species for which the kinetics of GTP-dependent activation have been examined, the \(K_{\rm A}\) values range from 1.6 uM to \(\sim\)250 uM (Table 2). Compared to the intracellular concentrations of GTP, which are typically 468 \(\pm\) 224 uM [112], it appears that CTPSs would be operating at near maximal activation by GTP under typical physiological conditions. Since intracellular concentrations of GTP in normal human cells and human tumor cells are 232 \(\pm\) 202 uM and 473 \(\pm\) 214 uM [112], respectively, this appears to be especially true for hCTPS1 with the lowest \(K_{\rm A}\) value reported to date [100]. However, the following observations present a possible caveat to this generalization. Using a mutant mouse T lymphoblast cell line (FURT-1A), which contained a CTPS refractory to complete inhibition by CTP, [PERSON] and [PERSON] [103] showed that exogenous manipulation of levels of GTP between 10% and 200% of that of unperturbed cells by either mycophenolic acid or guanosine revealed that the cooperativity of the enzyme with respect to GTP activation was much greater in situ (\(n\) = 1.59) than in vitro (\(n\) = 0.76). Furthermore, the concentrations of GTP required to activate the enzyme in situ were much _greater_ than those in vitro.