## 2 Regulation of CTPS Activity by GTP
### Structure-Activity Studies
#### 2.3.1 The L13 Loop: Limited Proteolysis  
Using trypsin-catalyzed proteolysis of _Ec_CTPS, [PERSON] and co-workers showed that Arg 429 and Lys 432 (Table 1) reside on an exposed loop (L13, Figure 2) [81]. Subsequent site-directed mutagenesis studies revealed that R429A _Ec_CTPS exhibited wild-type levels of NH\({}_{3}\)-dependent CTP formation; however, the values of \(k_{\text{cat}}\) and \(k_{\text{cat}}/K_{\text{m}}\) for Gln-dependent CTP formation were reduced \(\sim\)10- and \(\sim\)20-fold, respectively, relative to the wild-type enzyme. The \(K_{\text{m}}\) for Gln was not significantly altered. Activation of the glutaminase activity was reduced 6-fold at saturating concentrations of GTP, and the GTP-binding affinity was reduced 10-fold. These observations indicated that Arg 429 plays a role in both GTP-dependent allosteric activation of Gln hydrolysis and GTP binding [81]. Figure 2: Amino acid sequence alignment of selected CTP synthase proteins. Regions of the primary amino acid sequence corresponding to the N-terminal synthase (amidoligase) domain, the interdomain  
linker, and the C-terminal GAT domain are shaded light blue, light purple, and pink, respectively. Residues comprising the Cys-Glu-His triad are highlighted in red and the residues comprising the gate at the distal end of the NH\({}_{3}\) tunnel are highlighted in dark blue. Residues highlighted in light green, dark green, cyan, and purple play a role in GTP-dependent activation and correspond to the similarly colored residues in the structure shown in Figure 3A. The secondary structural assignments are those proposed by [PERSON] and co-workers for _Ec_CTPS [32; 44]. The species include: _Escherichia coli_ (_Ec_, P0A7E5), _Lactococcus lactis_ (_Li_, Q87761), _Thermus thermophilus_ (_Tt_, Q72 IN0), _Mycobacterium tuberculosis_ (_Mt_, P9 WHK7), _Saccharomyces cerevisiae_ (_Sc_, P28274, URA7), _Drosophila melanogaster_ (_Dm_, Q9 VUL1), and _Homo sapiens_ (_Hs_, P17812, CTPS1). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Variant** & **Organism** & \multicolumn{4}{c}{**Observations**} \\  & & **(Changes in Kinetic Parameters Are Relative to Wild-Type Enzyme)** & & \\ \hline \multicolumn{2}{c}{**(_E. coli Residue)**,} & & **NH\({}_{3}\)-Dependent** & **GTP-Dependent** & **Glutaminase Activity** & \\ \multicolumn{2}{c}{**Structural**} & & **CTP Formation** & **Gln-Dependent** & **in the Presence** & **Reference** \\ \multicolumn{2}{c}{**Element a**} & & & **CTP Formation** & **of GTP** & \\ \hline R105A, L4 & _E. coli_ & \begin{tabular}{c} no change in \(k_{\text{cat}}\) \\ no change in \(K_{\text{m}}\) \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 4.0-fold & \(\downarrow\)\(k_{\text{cat}}\) 2.5-fold & [82] \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.2-fold & \(\uparrow\)\(K_{\text{A}}\) 3.1-fold & \\ \hline D107A, L4 & _E. coli_ & \begin{tabular}{c} no change in \(k_{\text{cat}}\) \\ \(\downarrow\)\(k_{\text{cat}}\) 1.2-fold \\ \(\downarrow\)\(K_{\text{m}}\) 1.3-fold \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 1.7-fold & \(\downarrow\)\(k_{\text{cat}}\) 1.2-fold & [82] \\ \multicolumn{2}{c}{} & & \(\downarrow\)\(K_{\text{m}}\) 1.3-fold & \(\uparrow\)\(K_{\text{A}}\) 1.6-fold & \\ \hline L109A, L4 & _E. coli_ & \begin{tabular}{c} \(\downarrow\)\(k_{\text{cat}}\) 1.2-fold \\ \(\downarrow\)\(K_{\text{m}}\) 1.3-fold \\ \end{tabular}  & \(\downarrow\)\(K_{\text{cat}}\) 3.7-fold & no change in \(k_{\text{cat}}\) & [82] \\ \multicolumn{2}{c}{} & & \(\downarrow\)\(K_{\text{m}}\) 1.2-fold & \(\uparrow\)\(K_{\text{A}}\) 3.8-fold & \\ \hline L109A, L4 & _E. coli_ & \begin{tabular}{c} no change in \(k_{\text{cat}}\) \\ no change in \(K_{\text{m}}\) \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 7.2-fold b & \(\downarrow\)\(k_{\text{cat}}\) 3.0-fold b & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.5-fold b & \(\downarrow\)\(K_{\text{m}}\) 1.2-fold b & \\ \hline L109F, L4 & _E. coli_ & \begin{tabular}{c} \(\uparrow\)\(k_{\text{cat}}\) 1.2-fold \\ no change in \(K_{\text{m}}\) \\ \end{tabular}