Kinetic studies on wild-type and mutant Escherichia coli cytidine 5' -triphosphate synthases and inhibitor development

Cytidine 5'-triphosphate (CTP) synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or L-glutamine (Gln) as the source of nitrogen. CTPS catalyzes the hydrolysis of Gln within its C-terminal Gln amide transfer (GAT) domain and then transfers NH 3 to the active site of its N-terminal synthase domain, to better understand the mechanism of action for CTPS, a number of site-directed mutagenesis studies were conducted, these studies suggest that a mutation of Leu 109 to alanine causes a constriction within the NH3 tunnel, that Lys 297 is an absolutely critical residue for GTP-dependent activation of CTPS, that Arg 429 plays an important role in both GTP-dependent activation and GTP binding, that Lys 306 interacts with ATP, and that Gly 142 is a "hot spot" for nucleotide-dependent oligomerization required for conversion of CTPS to active tetramers.;When Gln is the substrate, GTP is required as a positive allosteric effector to promote Gln hydrolysis. However, GTP concentrations > 0.15 mM inhibit Gln-dependent CTP formation but not the intrinsic glutaminase activity of CTPS. In addition, GTP inhibits NH3-dependent CTP formation in a concentration-dependent manner, to delineate the structural determinants required for GTP-dependent activation and inhibition of CTPS, a structure-activity study was conducted using a variety of GTP and guanosine analogues. This study revealed that the requirements for activation are quite stringent with only a few GTP analogues capable of activating Gln-dependent CTP formation to varying degrees: GTP ≈ 6-thio-GTP > ITP ≈ guanosine 5'-tetraphosphate > O 6-methyl-GTP > 2'-deoxy-GTP. Inhibition of both NH3- and Gln-dependent CTP formation appeared to arise solely due to the purine base and was relatively insensitive to the identity of the purine with the exception of the requirement of an amino or carbonyl group at the 2-position. Caffeine, caffeine derivatives, uric acid derivatives, as well as phosphonate/phosphoramidate-based transition state and multivalent analogues were also shown to be inhibitors of CTPS.