Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax

Abstract

Cytidine 5′-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or L-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH3-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP ≈ 6-thio-GTP > ITP ≈ guanosine 5′-tetraphosphate > O6-methyl-GTP > 2′-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2′,3′-dideoxy- GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5′-triphosphate, 2′-OH, and 3′-OH are required for full activation. The 2-NH2 group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH2 group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH 3- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC50 ≈ 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC50 ≈ 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC50 = 80 μM). Ourfindingssuggestthatmodifying2-aminopurineor2-aminopurine riboside may serve as an effective strategy for developing cytidine 5′-triphosphate synthase inhibitors. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.