## 2 Regulation of CTPS Activity by GTP
### Structure-Activity Studies
#### 2.3.2 The L4 Loop  
[PERSON] and [PERSON] [82] conducted scanning alanine mutagenesis on a highly conserved region between residues 102 and 118 in _E_cTPS (shaded light green in Figure 2) and showed that residues located centrally within this region functioned to ensure efficient coupling of Gln-dependent NH\({}_{3}\) formation to the synthase activity. Significantly, the D107A and L109A variants (Table 1) exhibited wild-type levels of Gln hydrolysis and NH\({}_{3}\)-dependent CTP formation; however, Gln-dependent CTP formation was markedly impaired. Interestingly, \(K_{\rm A}\) for GTP binding, the GTP-dependent glutamine activity, the \(K_{\rm m}\) for Gln, and UTP- and ATP-dependent tetramerization were not altered significantly  
\begin{table}
\begin{tabular}{c} \% filaments, average \\ filament length, and median \\ filament length: 54\%, \\ 0.83 \(\upmu\)m, and 0.79 \(\upmu\)m \\ \end{tabular} & NR & [58] \\ \hline \hline \end{tabular}  
* As defined for _E_cTPS in [32], b CH-OH as the substrate [83]; c estimates only since determination was limited for technical reasons [84]; d value is an apparent value since the [Gln] may not be saturating under the experimental conditions [84]. e in the presence of ATP\(\gamma\)S (1 mM) and UTP (1 mM) [84]; f NR = not reported; 8 compared with wild-type enzyme where the % filaments, average filament length, and median filament length were 69\%, 1.05 \(\upmu\)m, and 1.06 \(\upmu\)m, respectively [58]. Arrows indicate the direction of the change.  
\end{table}
Table 1: _Cont._by the amino acid substitutions. These observations suggested that the D107A and L109A substitutions altered the normal structure of the NH\({}_{3}\) tunnel (i.e., the tunnel is leaky or constricted) or caused a structural perturbation that prevented the formation of a functioning tunnel, the effect being more pronounced for the L109A variant [82]. To test the hypothesis that the L109A substitution was in some way constricting the NH\({}_{3}\) tunnel, [PERSON] and [PERSON] [83] compared the kinetic parameters of Gln and NH\({}_{3}\) with those of the corresponding bulkier substrates L-\(\gamma\)-glutamyl hydroxamate (Gln-OH) and NH\({}_{2}\)OH, as well as the coupling ratios (Equations (4) and (5)) observed for Gln and Gln-OH with the wild-type and L109A _Ec_CTPSs. Interestingly, the L109A variant exhibited greater uncoupling with the bulkier nascent NH\({}_{2}\)OH, derived from Gln-OH hydrolysis, than with NH\({}_{3}\) derived from Gln hydrolysis. These observations were consistent with the L109A variant possessing a constricted or malformed NH\({}_{3}\) tunnel.  
\[\text{sub}-\text{saturating~{}cooling~{}ratio}=\frac{(k_{\text{cat}}/K_{\text {m}})_{\text{CTP~{}formation}}}{(k_{\text{cat}}/K_{\text{m}})_{\text{glutaminase~{} activity}}} \tag{4}\]  
\[\text{saturating~{}cooling~{}ratio}=\frac{(k_{\text{cat}})_{\text{CTP~{} formation}}}{(k_{\text{cat}})_{\text{glutaminase~{}activity}}} \tag{5}\]  
These authors also examined the kinetic properties of the L109F variant to see if the NH\({}_{3}\) tunnel could be fully blocked. This substitution, however, yielded a marked reduction in the glutamine activity arising from either a local perturbation at the Gln site such that Gln binding was not affected but its rate of hydrolysis was reduced, or a failure of GTP binding to induce the appropriate conformational change that promotes hydrolysis of Gln. The authors favored the latter scenario since the \(K_{\text{A}}\) value for GTP binding was similar to that of the L109A variant, indicating that the loss of glutaminase activity did not arise from impaired binding of GTP. A similar kinetic profile for the R105A variant also suggested that Arg 105 was required for efficient Gln turnover [82].