Cytidine 5-triphosphate synthase catalyzes the ATPdependent formation of CTP from UTP using either NH 3 or L-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Glndependent CTP formation at concentrations of >0.15 mM and NH 3 -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 > O 6 -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-NH 2 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-NH 2 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 (IC 50 ≈ 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 (IC 50 ≈ 4 -12 mM). 8-Oxoguanosine was the best inhibitor identified (IC 50 ‫؍‬ 80 M). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5-triphosphate synthase inhibitors.CTP synthase (CTPS 2 ; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)) catalyzes the ATP-dependent formation of CTP from UTP using either L-glutamine (Gln) or NH 3 as the nitrogen source (Scheme 1) (1, 2). This glutamine amidotransferase is a single polypeptide chain consisting of two domains. The C-terminal Gln amide transfer domain catalyzes the hydrolysis of Gln. The nascent NH 3 derived from this glutaminase activity is transferred via an NH 3 tunnel (3) to the N-terminal synthase domain, where it reacts with UTP that has been activated by ATP-dependent phosphorylation at the 4-position (4 -7). CTPS from Escherichia coli is the most thoroughly characterized CTPS with respect to its physical, kinetic, and structural properties. Indeed, E. coli CTPS serves as a good model for understanding catalysis by other CTPSs, including human CTPS (8), since CTPSs exhibit high conservation of functionally and structurally important residues and have few insertion/ deletion differences (3, 9).The de novo biosynthesis of pyrimidine nucleotides is highly regulated in E. coli, and consequently, CTPS is regulated in a complex fashion (1). GTP is required as a positive allosteric effector to increase the efficiency (k cat /K m ) of Gln-dependent CTP synthesis (10) by stabilizing the enzyme conformation that binds the tetrahedral intermediates formed during Gln ...

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.

Cytidine 5¢-triphosphate (CTP) synthase catalyses the ATPdependent formation of CTP from uridine 5¢-triphosphate using either NH 3 or L-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH 3 that is generated is transferred via an NH 3 tunnel [Endrizzi, J.A., Kim CTP synthases to utilize NH 3 , NH 2 OH, and NH 2 NH 2 as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, c-glutamyl hydroxamate, and c-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of c-glutamyl hydroxamate and nascent NH 2 OH production from N 4 -hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH 3 tunnel of L109A, in the presence of bound allosteric effector guanosine 5¢-triphosphate, is not leaky but contains a constriction that discriminates between NH 3 and NH 2 OH on the basis of size.Keywords: amidotransferase; ammonia tunnel; CTP synthase; glutaminase; alternative substrates.Cytidine 5¢-triphosphate (CTP) synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine (Gln) or NH 3 as the nitrogen source [1,2]. This Gln amidotransferase is a single polypeptide chain consisting of two domains. The C-terminal Gln amide transfer (GAT) domain utilizes a Cys-His-Glu triad to catalyse the rate-limiting hydrolysis of Gln (glutaminase activity) [3][4][5], and the nascent NH 3 derived from this glutaminase activity is transferred to the N-terminal synthase domain where the amination of a phosphorylated UTP intermediate is catalysed [6,7]. The reactions catalysed by CTPS are summarized in Scheme 1.CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. As CTP has a central role in the biosynthesis of nucleic acids [8] and membrane phospholipids [9], CTPS is a recognized target for the development of antineoplastic agents [8,10], antiviral agents [10][11][12], and antiprotozoal agents [13][14][15]. The Escherichia coli enzyme is one of the most thoroughly characterized CTP synthases with respect to its physical and kinetic properties, and is regulated in a complex fashion [1]. GTP is required as a positive allosteric effector to increase the efficiency of the glutaminase activity and Gln-dependent CTP synthesis [3,16] but inhibits CTP synthesis at concentrations > 0.15 mM [17]. In addition, the enzyme is inhibited by the product CTP [18] and displays positive cooperativity for ATP and UTP [18][19][20]. ATP and UTP act synergistically to promote tetramerization of the enzyme to its active form [20].Recently, the X-ray crystal structure of E. coli CTPS was solved at a resolution of 2.3 Å [21]. The enzyme crystallised as a tetramer, presumably because of the high protein concentrations used as bound nucleotides were not present in the structure (i.e. apo-E. coli CTPS) [21]. The authors identified a solvent-fi...

Cytidine 5¢-triphosphate synthase catalyses the ATPdependent formation of CTP from UTP using either ammonia or L-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/ K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH 3 -dependent CTP formation. For these mutants, the values of k cat /K m and k cat for glutamine-dependent CTP formation were reduced % 20-fold and % 10-fold, respectively, relative to wild-type enzyme; however, the value of K m for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.Keywords: activation; amidotransferase; CTP synthase; glutaminase; proteolysis; site-directed mutagenesis.CTP synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine or NH 3 as the nitrogen source (Scheme 1) [1,2]. This glutamine amidotransferase is a single polypeptide chain containing 545 amino acids and consisting of two domains. The C-terminal glutamine amide transfer (GAT) domain catalyses the hydrolysis of glutamine, and the nascent NH 3 derived from glutamine hydrolysis is transferred to the N-terminal synthase domain where the amination of UTP is catalysed [3,4]. CTPS belongs to the Triad family of glutamine amidotransferases [5,6] which utilizes a Cys-His-Glu triad to catalyse glutamine hydrolysis and also includes anthranilate synthase, carbamoyl phosphate synthase, formylglycinamidine synthase, GMP synthase, imidazole glycerol phosphate synthase, and aminodeoxychorismate synthase.CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. Because CTP has a central role in the biosynthesis of nucleic acids [7] and membrane phospholipids [8], CTPS is a recog...

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