## 2 Regulation of CTPS Activity by GTP
### Structure-Activity Studies
#### 2.3.1 The L13 Loop: Limited Proteolysis
 & \(\downarrow\)\(k_{\text{cat}}\) 5.9-fold & \(\downarrow\)\(k_{\text{cat}}\) 5.8-fold & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{A}}\) 4.9-fold & \\ \hline R429A, L13 & _E. coli_ & \begin{tabular}{c} no change in \(k_{\text{cat}}\) \\ no change in \(K_{\text{m}}\) \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 15-fold & \(\downarrow\)\(k_{\text{cat}}\) 6.0-fold & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.7-fold & \(\uparrow\)\(K_{\text{A}}\) 12-fold & \\ \hline G352P, L11 & _E. coli_ & \begin{tabular}{c} \(\downarrow\) specific activity \\ 1.3-fold \\ \end{tabular}  & no activity observed & – & [31] \\ \hline R359M & _L. lactis_ & \begin{tabular}{c} no change in \(k_{\text{cat}}\) \\ no change in \(K_{\text{m}}\) \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 12-fold & \(\downarrow\)\(k_{\text{cat}}\) 1.3-fold & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.7-fold & \(\uparrow\)\(K_{\text{A}}\) 4.6-fold & \(\uparrow\)\(K_{\text{m}}\) 1.2-fold & \\ \hline R359P & _L. lactis_ & \begin{tabular}{c} \(\downarrow\)\(k_{\text{cat}}\) 1.2-fold \\ \(\uparrow\)\(K_{\text{m}}\) 1.7-fold \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 43-fold & \(\downarrow\)\(k_{\text{cat}}\) 4.3-fold & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.7-fold & \(\uparrow\)\(K_{\text{A}}\)\(\sim\)11-fold c & no change in \(K_{\text{m}}\) & [84] \\ \hline G360A & _L. lactis_ & \begin{tabular}{c} \(\uparrow\)\(k_{\text{cat}}\) 1.2-fold \\ no change in \(K_{\text{m}}\) \\ \end{tabular}  & \(\uparrow\)\(k_{\text{cat}}\) 1.3-fold & \(\uparrow\)\(K_{\text{m}}\) 1.2-fold & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{cat}}\) 1.6-fold e & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{cat}}\) 1.6-fold e & \\ \multicolumn{2}{c}{} & & \(\downarrow\) no change in \(K_{\text{A}}\) e & \\ \hline G360P & _L. lactis_ & \begin{tabular}{c} \(\downarrow\)\(k_{\text{cat}}\) 3.5-fold \\ \(\uparrow\)\(K_{\text{m}}\) 1.7-fold \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) -24-fold c & – & [84] \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.7-fold & \(\uparrow\)\(K_{\text{A}}\) 142-fold c & – & [84] \\ \hline E362Q & _L. lactis_ & \ \begin{tabular}{c} \(\uparrow\)\(k_{\text{cat}}\) 1.6-fold \\ \(\uparrow\)\(K_{\text{m}}\) 1.4-fold \\ \end{tabular}  & \(\uparrow\)\(k_{\text{cat}}\) 1.2-fold & \(\downarrow\)\(k_{\text{cat}}\) 4.3-fold & \\ \multicolumn{2}{c}{} & & \(\uparrow\)\(K_{\text{m}}\) 1.4-fold & no change in \(K_{\text{A}}\) & no change in \(K_{\text{m}}\) & [84] \\ \hline T431V & _L. lactis_ & \begin{tabular}{c} \(\uparrow\)\(k_{\text{cat}}\) 1.2-fold \\ \(\uparrow\)\(K_{\text{m}}\) 1.7-fold \\ \end{tabular}  & \(\downarrow\)\(k_{\text{cat}}\) 104-fold & \(\downarrow\)\(k_{\text{cat}}\) 2.8-fold & \\ \multicolumn{2}{c}{} & & \(\downarrow\)\(k_{\text{cat}}\) 104-fold & no change in \(K_{\text{m}}\) & no change in \(K_{\text{m}}\) & [85] \\ \multicolumn{2}{c}{} & & & \(\downarrow\)\(k_{\text{cat}}\) 2.6-fold & \\ \multicolumn{2}{c}{} & & \(\downarrow\)\(k_{\text{cat}}\) 12.7-fold & \(\downarrow\)\(k_{\text{cat}}\) 1.6-fold & \\ \multicolumn{2}{c}{} & & \(\downarrow\)\(K_{\text{m}}\) 17-fold &Arg 429 and Lys 432 lie close to a conserved sequence motif [GG(TS)(ML)RLG)] (shaded purple in Figure 2) within the GAT domain that [PERSON] suggested played a role in GTP-dependent activation [85]. This suggestion was based on the observation that the motif was only present in CTPSs and not in the GAT domain of other class I Gln-dependent amidotransferases, which are not regulated by GTP binding. Through site-directed mutagenesis and kinetics studies, [PERSON] demonstrated that Thr 431 and Arg 433 within this motif of _L_ICTPS (Thr 438 and Arg 440 in _E_cTPS) play a role in GTP-dependent activation of Gln hydrolysis [85]. Since the T431V _L_ICTPS variant exhibited wild-type affinity for GTP, it was concluded that the hydroxyl group of Thr 431 plays a role only in the structural changes accompanying GTP-dependent activation.