Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate γ-semialdehyde. Glutamate γ-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate γ-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (Kis 0.16±0.03mM; Kii 0.4±0.1mM) and a competitive inhibitor with respect to ammonia (Ki 0.39±0.06mM) in the presence of GTP at pH8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate γ-semialdehyde. Although glutamate γ-semialdehyde exists in solution primarily in its cyclic form, Δ1-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Δ1-pyrroline-5-carboxylate (l-proline, l-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate γ-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

Abbreviations used: CPS, carbamoyl phosphate synthase; DON, 6-diazo-5-oxonorleucine; GAT, glutamine amide transfer; GlmS, glucosamine-6-phosphate synthase; GSA, glutamate γ-semialdehyde; His6-CTPS, CTP synthase containing an N-terminal hexahistidine tag; P5C, Δ1-pyrroline-5-carboxylate.

This content is only available as a PDF.