## 2 Regulation of CTPS Activity by GTP
### Regulatory Effects of NADH
The cleft is formed by residues 50-55, 297-301, 353-356, 438-441, 468, and 470. [PERSON] and co-workers constructed a model of GTP bound at this site (Figure 3A) [32], which has permitted the results from site-directed mutagenesis and kinetics experiments to be put into context. Indeed, this binding site has now been confirmed with the first structure of CTPS with GTP bound (vide infra) [66]. Figure 3: Conservation, electrostatics, and interacting residues of the GTP-binding site. (**A**) Sterewoview of the GTP-binding site (sphere representation) is shown with the same orientation as in panels B and C. GTP (space-filling representation) is shown as modeled into the cleft of _Ec_CTPS (PDB ID: 1S1M) by [PERSON] and co-workers [32; 44] with those residues implicated in GTP-dependent activation of CTPS (Table 1) highlighted: residues of the 104-110 loop (light green; especially Asp 107 and Leu 109), residues 352–360 of the L11 lid (dark green; especially Gly 352, Arg 356, and Gly 360), residues of the 429–440 loop (purple; especially Arg 429, possibly Lys 432, Thr 438, and Arg 440), residues Glu 68, Arg 468, Arg 470, and Thr 51 (cyan), and Met 52 (yellow). The coloring of the amino acid residues corresponds to the shading of the amino acids in the sequence alignment shown in Figure 2. The light green and magenta arrows suggest movement of the similarly colored residues upon GTP binding. Clearly, the enzyme must undergo a change in conformation (suggested by arrows) from that shown so that the protein interacts with the 6-position of the purine ring since GTP-dependent activation of Gln-dependent CTP formation requires the presence of a carbonyl group at the 6-position of GTP. (**B**) Conservation of residues based on a multiple sequence alignment prepared and rendered in surface representation using the CONSURF server [88] is shown for two chains of the CTPS tetramer. Highly conserved residues (purple) line the GTP-binding cleft denoted by the purple arrow. (**C**) Electrostatic potential surface of the CTPS tetramer (surface representation) is shown with the same orientation as in panel B. The blue arrow denotes the electropositive region that binds the 5′-triphosphate moiety of GTP. The GTP-binding site resides in a region of highly conserved amino acid sequence (Figure 3B), which exhibits a predominantly negative electrostatic potential surface for interacting with the 5\({}^{\prime}\)-triphosphate moiety of GTP (Figure 3C). With the exception of the amino acid residues on the flexible loop L13 between Val 428 and Thr 438, all the conserved residues listed in Table 1 that were implicated in the GTP-dependent activation of Gln-dependent CTP formation by site-directed mutagenesis experiments line this cleft (Figure 3A). In the structure, loop L4 (_Ec_CTPS residues 105-114) resides between the GAT and synthase active sites and forms part of the rim of the GTP-binding site. Arg 468, Arg 470, and Glu 68 appear to interact with the purine moiety of GTP [32]. Significantly, Leu 109 is located on a flexible loop of an adjacent subunit, which extends over the deep cleft separating the GAT and synthase sites. This places Leu 109 above the opening that [PERSON] et al. [32] had identified as a putative entry point for NH\({}_{3}\) to access the solvent-filled \"vestibule\" connecting the GAT active site and the GAT/syntase interface. Leu 109 is not interacting with GTP in the model. However, the kinetic properties of the L109A _Ec_CTPS variant suggested that the residue is involved with GTP-dependent activation of the enzyme. Clearly, Leu 109 does not directly cause a constriction in the NH\({}_{3}\) tunnel, but substitution by Ala alters the kinetics by interfering with the appropriate conformational changes that accompany GTP binding and proper formation of the NH\({}_{3}\) tunnel. As such, the resulting tunnel may still be constricted or malformed. [PERSON] and co-workers suggested that a polypeptide linker connecting the L4 loop and the symmetry-related subunit could mediate coupling between ATP-binding and Gln hydrolysis [44].