The Subunit Structure and Subunit Interactions of Cytidine Triphosphate Synthetase

Inhibition of Escherichia coli CTP synthase by glutamate γ-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis

“…Our observations indicate that GSA competitively inhibits the ammonia-dependent activity of CTP synthase by binding at a distinct glutamine site whose affinity for glutamine is altered by the presence of GTP. It seems unlikely that the binding of GSA and the formation of the thiohemiacetal sterically blocks access to the ammonia site because covalent modification by the glutamine analogue 6-diazo-5-oxonorleucine (DON), which is larger than either glutamine or GSA by a methylene group, does not block access to the ammonia site [15]. A more plausible explanation is that reaction of GSA at the glutamine site to form the thiohemiacetal induces a conformational change that prevents exogenous ammonia from reaching its site of reaction in a manner similar to that of either glutamylated enzyme or the tetrahedral intermediates generated during glutamine hydrolysis (Scheme 4B).…”

“…A more plausible explanation is that reaction of GSA at the glutamine site to form the thiohemiacetal induces a conformational change that prevents exogenous ammonia from reaching its site of reaction in a manner similar to that of either glutamylated enzyme or the tetrahedral intermediates generated during glutamine hydrolysis (Scheme 4B). Such major conformational changes are induced by DON and glutamine [15,37] and are believed to give rise to the ' half-of-the-sites ' reactivity observed when DON reacts with only half of the total glutamine sites yet abolishes all of the glutamine activity of the enzyme and leaves the ammonia activity intact [37]. Distinct differences between the native, glutamylated and DON-modified enzyme have been demonstrated by their differing reactivities towards 5,5h-dithiobis-(2-nitrobenzoic acid) [37].…”

“…In the sequence of reactions catalysed by CTP synthase, glutamine is first hydrolysed to yield glutamate and nascent ammonia. The GAT domain of E. coli CTP synthase contains a reactive cysteine residue at position 379 that is believed to participate in the formation of a γ-glutamyl S-ester intermediate, as shown in Scheme 2(A) [2,4,15,16]. The nascent ammonia generated via glutamine hydrolysis reacts with UTP that has been activated by phosphorylation at the 4-position, to yield CTP [15,17].…”

“…The GAT domain of E. coli CTP synthase contains a reactive cysteine residue at position 379 that is believed to participate in the formation of a γ-glutamyl S-ester intermediate, as shown in Scheme 2(A) [2,4,15,16]. The nascent ammonia generated via glutamine hydrolysis reacts with UTP that has been activated by phosphorylation at the 4-position, to yield CTP [15,17]. GTP is an allosteric effector that stimulates the enzyme's glutaminase activity but has no effect on the reaction when ammonia is the substrate [18].…”

Inhibition of Escherichia coli CTP Synthetase by NADH and Other Nicotinamides and Their Mutual Interactions with CTP and GTP

“…All four nucleotides contribute activating or inhibiting inputs. Active tetramer assembly from inactive dimers is promoted by ATP and UTP substrates, − which bind at an interfacial active site formed by the N-terminal amidoligase domains. ,, Feedback inhibition is effected by the product CTP, which competes with UTP by binding at a distinct but overlapping site . Allosteric regulator GTP is required for efficient CTP synthesis but also can inhibit it; low concentrations (0–200 μM) activate glutamine hydrolysis up to 50-fold, while higher concentrations (IC 50 ≈ 500 μM) inhibit CTP synthesis without affecting glutamine hydrolysis .…”