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Significance In the current study, we successfully push the resolution to near-atomic levels (2.48 Å) to analyze the polymer structure of Drosophila melanogaster cytidine triphosphate synthase (dmCTPS) with all its substrates (6-diazo-5-oxo-L-norleucine being used to represent glutamine). We have precisely located all ligands in a CTPS structure with a solid electron density map. With this model, we present a structural conformation of the GTP binding site and demonstrate its roles in mediating glutamine binding, NH3 transport, and stabilizing the ammonia tunnel. Additionally, the intermediate in the ATP-dependent phosphorylation reaction is observed allowing us to identify the residues participating in catalysis., Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo–electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

In biology, supramolecular recognition typically involves an 'induced‐fit' mechanism, where structures rearrange upon complexation to accommodate binding ligands. Designing minimalistic compounds with such adaptability is challenging as they involve subtle conformational changes that are energetically similar. Here, we demonstrate the integration of combinatorial screening with molecular modelling to identify heptapeptides that form a stable loop upon recognition of uridine triphosphate (UTP). Peptide sequences selected using phage display were refined computationally and correlated with experimental KD values. This combined approach may serve as a method for the de novo selection and subsequent rationalization of the compositional and organizational principles that dictate chemical functionality in flexible structures with dynamic conformations.

Nucleotide-binding proteins play important roles in a variety of biological processes. While ATP- and GTP-binding proteins have been well studied, the systematical identification of UTP-interacting proteins remains under investigated. Here, we developed a chemical proteomic strategy using a biotinylated UTP affinity probe coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) method to enrich, identify and quantify UTP-binding proteins at the entire proteome scale. By performing labeling reactions with high vs low concentrations of UTP probe (100 and 10 μM) or with the UTP probe in the presence of free UTP in stable isotope labeling by amino acids in cell culture (SILAC) experiments, we identified more than 70 potential UTP-binding proteins which are involved in multiple cellular processes, such as translational elongation and protein folding. We also validated the UTP-binding capability of the cytoskeletal protein ACTB by using cellular thermal shift assay (CETSA). Together, we performed a high-throughput chemical proteomics-based analysis to identify, for the first time, UTP-binding proteins in human proteome, which should be applicable for the identification and quantification of UTP-binding proteins in other organisms.

The active metabolite GS-461203 of hepatitis C virus (HCV) non-structural protein 5B (NS5B) inhibitor sofosbuvir can stall RNA synthesis or replication by competitively inhibiting the natural substrate nucleoside triphosphate like UTP. Unfortunately, S282T mutant can lead to the resistance to sofosbuvir. Here, the detailed binding mechanism and unbinding process of GS-461203 and UTP to HCV NS5B were unraveled by using conventional molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation. Our simulation results demonstrate that both polar and nonpolar interactions are favorable for GS-461203 and UTP binding. Meanwhile, we also identified the key residues responsible for GS-461203 and UTP binding in NS5B-RNA together with the three unbinding process steps including translation, reversal of base and ribose and complete divorce. The 2′-fluoro-2′-C-methyl ribose of GS-461203 can form stronger polar and nonpolar interactions with residues S282 and I160 than UTP. The results can also explain the reason why GS-461203 can effectively be incorporated into RNA synthesis or replication. In the S282T mutant system, the binding affinity attenuation of UTP relative to wild type HCV NS5B is less than that of GS-461203. The obtained binding and unbinding mechanism of HCV NS5B with the inhibitor GS-461203 and substrate in our work will provide useful guidance for the development of new and effective HCV NS5B inhibitors with low resistance.

The causative agent of African sleeping sickness, Trypanosoma brucei , undergoes an unusual mitochondrial RNA editing process that is essential for its survival. RNA editing terminal uridylyl transferase 2 of T. brucei (TbRET2) is an indispensable component of the editosome machinery that performs this editing. TbRET2 is required to maintain the vitality of both the insect and bloodstream forms of the parasite, and with its high-resolution crystal structure, it poses as a promising pharmaceutical target. Neither the exclusive requirement of uridine 5'-triphosphate (UTP) for catalysis, nor the RNA primer preference of TbRET2 is well-understood. Using all-atom explicitly solvated molecular dynamics (MD) simulations, we investigated the effect of UTP binding on TbRET2 structure and dynamics, as well as the determinants governing TbRET2's exclusive UTP preference. Through our investigations of various nucleoside triphosphate substrates (NTPs), we show that UTP preorganizes the binding site through an extensive water-mediated H-bonding network, bringing Glu424 and Arg144 side chains to an optimum position for RNA primer binding. In contrast, cytosine 5'-triphosphate (CTP) and adenosine 5'-triphosphate (ATP) cannot achieve this preorganization and thus preclude productive RNA primer binding. Additionally, we have located ligand-binding "hot spots" of TbRET2 based on the MD conformational ensembles and computational fragment mapping. TbRET2 reveals different binding pockets in the apo and UTP-bound MD simulations, which could be targeted for inhibitor design.

The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60 kDa, exhibiting relatively high specific activity (∼80 Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15 μM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term. © 2008 Elsevier Masson SAS. All rights reserved. Fil: Bosco, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Machtey, Matías. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral; Argentina

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the first step in peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. The products of the GlmU reaction are essential for bacterial survival, making this enzyme an attractive target for antibiotic drug discovery. A series of Haemophilus influenzae GlmU (hiGlmU) structures were determined by X-ray crystallography in order to provide structural and functional insights into GlmU activity and inhibition. The information derived from these structures was combined with biochemical characterization of the K25A, Q76A, D105A, Y103A, V223A, and E224A hiGlmU mutants in order to map these active-site residues to catalytic activity of the enzyme and refine the mechanistic model of the GlmU uridyltransferase reaction. These studies suggest that GlmU activity follows a sequential substrate-binding order that begins with UTP binding noncovalently to the GlmU enzyme. The uridyltransferase active site then remains in an open apo-like conformation until N-acetylglucosamine-1-phosphate (GlcNAc-1-P) binds and induces a conformational change at the GlcNAc-binding subsite. Following the binding of GlcNAc-1-P to the UTP-charged uridyltransferase active site, the non-esterified oxygen of GlcNAc-1-P performs a nucleophilic attack on the alpha-phosphate group of UTP. The new data strongly suggest that the mechanism of phosphotransfer in the uridyltransferase reaction in GlmU is primarily through an associative mechanism with a pentavalent phosphate intermediate and an inversion of stereochemistry. Finally, the structural and biochemical characterization of the uridyltransferase active site and catalytic mechanism described herein provides a basis for the structure-guided design of novel antibacterial agents targeting GlmU activity.

Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [3H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[3H]inositol-loaded ampullas were determined. Specific [3H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 μM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/μg protein; dissociation constant ( KD1) = 0.4 μM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/μg protein; KD2= 10 μM). The stereospecificity pattern for UTP analog recognition was UMP > UDP ≥ ADP = UTP = dTTP > adenosine 5′- O-(3-thiotriphosphate) = ATP = CTP = 2′-and 3′- O-4-(benzoylbenzoyl)-ATP (Bz-ATP) ≥ AMP ≥ 2-methylthio-ATP = α,β-methylene-ATP > uridine = diadenosine tetraphosphate (Ap4A); cAMP and adenosine were inactive. Antagonist recognition pattern was DIDS = pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) = reactive blue 2 > suramin. The rank order of potencies for agonist-induced PLC activation was UDP ≥ UTP ≥ Ap4A ≥ UMP = Bz-ATP; uridine was inactive. UTP-stimulated PLC activity was inhibited by DIDS = reactive blue 2 = PPADS > suramin. These results suggest that the population of [3H]UTP-labeled binding sites is heterogeneous, with a low number of high-affinity UTP receptors whose function(s) need to be determined and a large number of moderate-affinity receptors triggering PLC activation.

It has recently been demonstrated that two types of ATP receptors, the P2Y and P2U receptors, are coexpressed on bovine aortic endothelial cells. The aim of the present study was to characterize directly P2Y and P2U subtypes on intact bovine aortic endothelial cells and on membranes prepared from these cells using adenosine 5'-0-(3-thio[35S]triphosphate) ([35S]ATP gamma S), [alpha-32P]ATP and [alpha-32P]UTP as radioligands. [35S]ATP gamma S binding to bovine aortic endothelial cell membranes was saturable and apparently involved a single class of high-affinity binding sites (Kd: 14 +/- 11 nM. Bmax 1.6 +/- 0.7 pmol/mg protein; mean +/- S.D.). A similar class of high-affinity binding sites was identified with [alpha-32P]ATP (Kd: 14 +/- 9 nM; Bmax: 1.7 +/- 1.1 pmol/mg protein; mean +/- S.D.). Competition experiments showed that only one third of these sites bound 2-methylthio-ATP (2-MeSATP) with high affinity (Ki: 21 +/- 5 and 14 +/- 10 nM, mean +/- S.D., for [35S]ATP gamma S and [alpha-32P]ATP, respectively) and might therefore represent the P2Y receptors. UTP did not compete with [35S]ATP gamma S or [alpha-32P]ATP for binding at the remaining sites, indicating that they are not the P2U receptors. No high-affinity UTP binding sites could be detected using [alpha-32P]UTP. [35S]ATP gamma S binding to intact bovine aortic endothelial cells was competed by ATP gamma S (Kd: 1.0 +/- 0.5 microM; mean +/- S.D.), but not by 2-MeSATP and UTP, indicating that these binding sites are neither the P2Y nor the P2U receptors.

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 A. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P ( N -acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure–activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery. Abbreviations: DTT, dithiothreitol; GlcNAc-1-P, N-acetylglucosamine-1-phosphate; GlcN-1-P, D-glucosamine-1-phosphate; HTS, high-throughput screen; ITC, isothermal titration calorimetry; MIC, minimum inhibitory concentration; PPi, pyrophosphate ion; RMSD, root mean square deviation; SAR, structure–activity relationship; UDP-GlcNAc, uridyldiphosphate-N-acetylglucosamine

In the nucleotide addition cycle, pyrophosphate is generated upon incorporation of each nucleotide. Rapid release of pyrophosphate is essential for facile transcription elongation. Stopped-flow kinetic studies involving alterations in the intrinsic protein fluorescence of the core polymerase upon the binding of pyrophosphate to well-defined elongation complexes (ECs) indicate that the intrinsic off-rate of pyrophosphate (k=5.7-8.1 s(-1)) is too slow to account for the rapid rate of nucleotide incorporation that occurs during processive transcription elongation. Stopped-flow kinetic studies on UTP binding followed by UMP incorporation into an EC as monitored by alterations in the intrinsic protein fluorescence of the core polymerase resulted in a set of first-order rate constants that varied in a hyperbolic manner as a function of UTP concentration. This is consistent with a binding step (K(UTP)=17±6 μM) followed by a conformational change (k=623±54 s(-1)) in the core polymerase. In comparable studies on ATP binding and AMP incorporation into an EC, the data were also consistent with a binding step (K(ATP)=44±6 μM) followed by a conformational change (k=411±51 s(-1)) in the core polymerase. In stopped-flow kinetic studies with α,β-methyleneadenosine 5' triphosphate, which can bind to the EC but cannot lead to nucleotide incorporation, the analysis of the hyperbolic dependence of the observed first-order rate constant on α,β-methyleneadenosine 5' triphosphate concentration yielded a value of 20±13 μM for the apparent dissociation constant and a value of 221±36 s(-1) for the first-order rate constant for the associated conformational change in the core polymerase. This indicates that the conformational change in the core polymerase precedes chemistry. In conjunction with previously reported results on the increase in the rate of pyrophosphate release in the presence of the next cognate nucleotide for incorporation, the data are consistent with a model in which rapid pyrophosphate release is coupled to a conformational change in the core polymerase that precedes chemistry and that occurs upon the binding of the next cognate nucleotide for incorporation.

RNA uridylylation reactions catalyzed by terminal uridylyl transferases (TUTases) play critical roles in the formation of the mitochondrial transcriptome in trypanosomes. Two mitochondrial RNA editing TUTases have been described: RNA editing TUTase 1 catalyzes guide RNA, ribosomal RNA, and mRNA 3'-uridylylation, and RNA editing TUTase 2 acts as a subunit of the RNA editing core complex (also referred to as the 20S editosome) to perform guided U-insertion mRNA editing. Although RNA editing TUTase 1 and RNA editing TUTase 2 carry out distinct functions and possess dissimilar enzymatic properties, their catalytic N-terminal domain and base recognition C-terminal domain display a high degree of similarity, while their middle domains are less conserved. MEAT1 (mitochondrial editosome-like complex associated TUTase 1), which interacts with an editosome-like assembly and is exclusively U-specific, nonetheless shows limited similarity with editing TUTases and lacks the middle domain. The crystal structures of apo MEAT1 and UTP-bound MEAT1 refined to 1.56 A and 1.95 A, respectively, reveal an unusual mechanism of UTP selection and domain organization previously unseen in TUTases. In addition to established invariant UTP-binding determinants, we have identified and verified critical contributions of MEAT1-specific residues using mutagenesis. Furthermore, MEAT1 possesses a novel bridging domain, which extends from the C-terminal domain and makes hydrophobic contacts with the N-terminal domain, thereby creating a cavity adjacent to the UTP-binding site. Unlike the minimal TUT4 TUTase, MEAT1 shows no appreciable conformational change upon UTP binding and apparently does not require RNA substrate to select a cognate nucleoside triphosphate. Because MEAT1 is essential for the viability of the bloodstream and insect forms of Trypanosoma brucei, the unique organization of its active site renders this protein an attractive target for trypanocide development.

The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves feedback inhibition by both CTP and UTP, although it is only in the presence of CTP that UTP appears to inhibit the activity of the enzyme. In order to better understand the parts played by both pyrimidine nucleotides in this synergistic inhibition, binding studies were performed by continuous-flow dialysis and ultracentrifugation methods. The results obtained show that UTP binds to ATCase in the absence of CTP. Nevertheless, this binding does not induce any inhibition unless CTP is present. The mutual influence of CTP and UTP on their respective binding constants suggests that they bind to the same regulatory sites. However, the results obtained cannot be satisfactorily explained by a simple competition between the nucleotides, and it is shown that reciprocal affinity enhancements play a fundamental role. CTP enhances the affinity of UTP for the regulatory sites 80-fold, and conversely, UTP enhances the affinity of CTP 5-fold. Interestingly, the isolated regulatory subunits bind the two pyrimidine nucleotides following the same pattern as the entire enzyme. These observations indicate that the synergistic inhibition mechanism relies entirely on interactions between the two adjacent allosteric sites which belong to the same regulatory dimer.

Escherichia coli aspartate carbamoyltransferase controls pyrimidine biosynthesis by feedback inhibition involving both CTP and UTP, although UTP only inhibits the enzyme in the presence of CTP (Wild, J. R., Loughrey-Chen, S. J., and Corder, T. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 46-50). The mechanism by which the enzyme can discriminate between these two pyrimidines is unknown, as well as where UTP binds and its mode of action. A mutant version of the enzyme with a single amino acid substitution in the regulatory site (Asp-19----Ala) causes loss of the synergistic inhibition of UTP in the presence of CTP, and furthermore, this enzyme is inhibited by UTP alone. Analysis of CTP binding to the mutant enzyme reveals that UTP can bind to the mutant enzyme in the absence of CTP but not in its presence. This is completely opposite to the wild-type enzyme in which case UTP only exhibits significant binding in the presence of CTP. Further analysis of the binding data for the wild-type enzyme reveals that, in the presence of UTP, CTP only binds to three sites, although CTP binds to six sites, three with high affinity and three with low affinity in the absence of UTP. Parallel UTP binding experiments in the presence of CTP suggest that UTP binds to the three weak CTP sites. The Asp-19----Ala substitution prevents UTP binding in the presence of CTP and allows UTP to bind and inhibit the enzyme in the absence of CTP. Since the x-ray data indicate no specific interactions between the amino group of cytosine and amino acid side chains in the regulatory binding site, the discrimination between UTP and CTP by the wild-type enzyme must be due to subtle differences in the binding sites rather than direct side chain contacts.

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of UTP. They are hexamers regulated by GTP (allosteric activator) and UTP (inhibitor). We describe here the 2.8 angstroms crystal structure of Escherichia coli UMP kinase bound to GTP. The GTP-binding site, situated at 15 angstroms from the UMP-binding site and at 24 angstroms from the ATP-binding site, is delineated by two contiguous dimers. The overall structure, as compared with those bound to UMP, UDP, or UTP, shows a rearrangement of its quaternary structure: GTP induces an 11 degrees opening of the UMP kinase dimer, resulting in a tighter dimer-dimer interaction. A nucleotide-free UMP kinase dimer has an intermediate opening. Superposition of our structure with that of archaeal UMP kinases, which are also hexamers, shows that a loop appears to hamper any GTP binding in archeal enzymes. This would explain the absence of activating effect of GTP on this group of UMP kinases. Among GTP-binding residues, the Asp-93 is the most conserved in bacterial UMP kinases. In the previously published structures of E. coli UMP kinase, this residue was shown to be involved in hydrogen bonds between the subunits of a dimer. Its substitution by an alanine decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition. This demonstrates that the previously described mutual exclusion of these two nucleotides is mediated by Asp-93.

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase P. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 angstrom crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 angstrom crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides. (c) 2006 Elsevier Ltd. All rights reserved.

Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447-6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface.

A non-steady-state kinetic method has been developed to observe the initiation of long RNA chains by Escherichia coli RNA polymerase without the enzyme turnover. This method was used to determine the order of binding of the first two nucleotides to the enzyme in RNA synthesis with the first two nucleotides to the enzyme in RNA synthesis with poly(dA-dT) as the template. It was shown that initiator [ATP, uridyly(3'-5')adenosine, or adenyly(3'-5')uridylyl-(3'-5')adenosine] binds first to the enzyme-template complex, followed by UTP binding. The concentration dependence of UTP incorporation into the initiation complex suggests that more than one UTP molecule may bind to the enzyme-DNA complex during the initiation process. Comparison of the kinetic parameters derived from these studies with those obtained under steady-state conditions indicates that the steps involving binding of initiator or UTP during initiation cannot be rate limiting in the poly(dA-dT)-directed RNA synthesis. The non-steady-state technique also provides a method for active-site titration of RNA polymerase. The results show that only 36 +/- 9% of the enzyme molecules are active in a RNA polymerase preparation of high purity and specific activity. In addition, the minimal length of poly(dA-dT) involved in RNA synthesis by one RNA polymerase molecule was estimated to be approximately 500 base pairs.

In all previous kinetics studies of calf liver CTP synthetase, simple Michaelis-Menten hyperbolic plots were obtained. In this study it was shown that calf liver CTP synthetase could generate sigmoidal kinetic plots as a function of the substrate UTP when in the presence of the product of the reaction, CTP. The Hill number was estimated to be 2.8. The enzyme did not generate sigmoidal plots as a function of the other substrates (L-glutamine and ATP) either in the presence or absence of CTP. Thus, CTP apparently induced changes in the liver enzyme which altered the binding of UTP to the enzyme by acting at a site distinct from the UTP binding site (allosteric site). This concept was further strengthened by the fact that 3-deazaUTP, a known competitive inhibitor of the liver enzyme, did not induce sigmoidal kinetic plots. It was also shown that CTP had no effect upon the dimerization of the enzyme, thus ruling out monomer to dimer transitions as a potential mechanism for the observed sigmoidal kinetics.

In Saccharomyces cerevisiae, the first two reactions of pyrimidine biosynthesis are catalyzed by the multifunctional protein Ura2 carrying both carbamyl-phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) enzyme activities. In order to study how UTP regulates both of these activities mutant strains were constructed: one strain which expressed the Ura2 protein fused to the green fluorescent protein, and two strains expressed truncated Ura2 proteins. These strains exhibited a phenotype associated with a modified regulation of the pyrimidine pathway. Results presented in this report provide arguments in favor of a single UTP binding site located on the CPSase domain, and support a model in which ATCase activity is inhibited by UTP only when it can interact with the CPSase domain.

The studies reported in this paper were undertaken to compare the steady-state kinetics of ATPase of purified platelet actomyosin and myosin free of actin. Actomyosin exhibits highly sigmoid kinetics with at least two interacting ATP or UTP binding sites. These studies were done at O.6 m KCl where actin and myosin are generally supposed to be dissociated in the presence of these nucleotides (M. Gallaghar, T. C. Detwiler, and A. Stracher, 1976, in Cell Motility (Goldman, R., Pollard, T., and Rosenbaum, J., eds.), Vol. 3, Part A, pp. 475–485 Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.; A. Weber, 1969, J. Gen Physiol. 53 , 781–791) . When the dissociation of platelet actomyosin was actually investigated by a sucrose density gradient technique under conditions which were similar to those of the steady-state kinetic experiments, only partial dissociation of actin from myosin was observed. This was especially true at low nucleotide concentrations where differences in the sigmoidicity of the saturation curves of actomyosin and actin-free myosin have been observed. These findings suggest that in platelet actomyosin, actin enhances the cooperativity of nucleotide binding sites of myosin by reducing the K m for ATP or UTP. In contrast, the saturation curves of platelet myosin using either ATP or UTP as substrates are less sigmoidal and possess an intermediary plateau region, when analyzed by Hill and reciprocal plots, these data indicate both positive and negative cooperativity suggesting more than two substrate binding sites. Platelet myosin also hydrolyzed other nucleotides (the order of rates being ITP > UTP > UTP > ATP > CTP > GTP). The kinetics of ITP differed from that of ATP or UTP in that no plateau region was observed on the saturation curve. In addition, no cooperativity of ITP binding sites was seen at low substrate concentrations (up to 0.2 m m ) but was instead observed at high ITP concentration. It is concluded that conformational changes in myosin induced by ITP may not be necessarily identical to those induced by ATP or UTP.