Kinetic characteristics and regulatory mechanisms of glucokinase and fructokinase from Zymomonas mobilis

Summary Glucokinase (EC 2.7.1.2) exhibits two pH optima (pH 7.0 and 8.2), gives hyperbolic saturation curves and reacts equally with ATP, UTP, GTP, ITP and CTP. Inhibition occurs with high concentrations of these nucleotides and in addition with ADP, AMP and glucose 6-phosphate. No inhibition was observed with sucrose, glucose, fructose (11 mM), ethanol (542 mM), mannose, ribose, galactose, deoxy-glucose, lactose and gluconate and no reaction except with glucose.Fructokinase (EC 2.7.1.4) exhibits one pH optimum (7.4), gives hyperbolic saturation curves and is highly specific towards ATP and fructose. Mannose, glucose, GTP and CTP do not react. Inhibition occurs with glucose, glucose 6-phosphate and mildly fructose 6-phosphate. ATP at high concentrations gives slight inhibition, ADP and AMP show differential effects, whereas all other above mentioned compounds do not inhibit.Regulatory mechanisms for sucrose, glucose and fructose metabolism are discussed.     

Selective high metabolic lability of uridine, guanosine and cytosine triphosphates in response to glucose deprivation and refeeding of untransformed and polyoma virus-transformed hamster fibroblasts.

Sugar deprivation of hamster fibroblasts (NIL) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the folloing fashion: the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma-transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar-supplemented medium.Refeeding glucose to sugar-depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.     

Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae

CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.     

ATP-dependent H+ transport in synaptosomal membrane vesicles of the rat brain

H+ transport into synaptosomal membrane vesicles of the rat brain was stimulated by ATP and to a lesser extent by GTP, but not by ITP, CTP, UTP, ADP, AMP or beta, gamma-methylene ATP. ATP at concentrations up to 200 mM concentration-dependently stimulated the rate of H+ transport with a Km value of 0.6 mM, but at higher concentrations of this nucleotide the rate decreased. Other nucleotides such as CTP, UTP, GTP and AMP, or products of ATP hydrolysis i.e. ADP and Pi also reduced the ATP-stimulated H+ transport. The inhibition by GTP and ADP was not affected by the ATP concentration. These findings suggest that plasma membranes of nerve endings transport H+ from inside to outside of the cells utilizing energy from ATP hydrolysis, and that this transport is regulated by the intracellular concentration of nucleotides and Pi on sites other than those involved in substrate binding.     

Characterization of nucleotide pools as a function of physiological state in Escherichia coli

Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a “rebound” of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.     

Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.     

Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP

Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent 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 a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH(3)-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme's intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH(3) as a substrate by the synthase domain.     

Differential effects of gemcitabine on ribonucleotide pools of twenty-one solid tumour and leukaemia cell lines

To gain a more detailed insight into the metabolism of 2', 2'-difluoro-2'-deoxycytidine (dFdC, gemcitabine, Gemzar) and its effect on normal ribonucleotide (NTP) metabolism in relation to sensitivity, we studied the accumulation of dFdCTP and the changes in NTP pools after dFdC exposure in a panel of 21 solid tumour and leukaemia cell lines. Both sensitivity to dFdC and accumulation of dFdCTP were clearly cell line-dependent: in this panel of cell lines, the head and neck cancer (HNSCC) cell line 22B appeared to be the most sensitive, whereas the small cell lung cancer (SCLC) cell lines were the least sensitive to dFdC. The human leukaemia cell line CCRF-CEM accumulated the highest concentration of dFdCTP, whereas the non-SCLC cell lines accumulated the least. Not only the amount of dFdCTP accumulation was clearly related to the sensitivity for dFdC (R=-0.61), but also the intrinsic CTP/UTP ratio (R=0.97). NTP pools were affected considerably by dFdC treatment: in seven cell lines dFdC resulted in a 1.7-fold depletion of CTP pools, in two cell lines CTP pools were unaffected, but in 12 cell lines CTP pools increased about 2-fold. Furthermore, a 1.6-1.9-fold rise in ATP, UTP and GTP pools was shown in 20, 19 and 20 out of 21 cell lines, respectively. Only the UTP levels after treatment with dFdC were clearly related to the amount of dFdCTP accumulating in the cell (R=0.64 (P<0.01)), but not to the sensitivity to dFdC treatment. In conclusion, we demonstrate that besides the accumulation of dFdCTP, the CTP/UTP ratio was clearly related to the sensitivity to dFdC. Furthermore, the UTP levels and the CTP/UTP ratio after treatment were related to dFdCTP accumulation. Therefore, both the CTP and UTP pools appear to play an important role in the sensitivity to dFdC.     

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.     

Fatty acid synthesis by isolated chromoplasts from the daffodil. Energy sources and distribution patterns of the acids

1. Fatty acid synthesis in isolated intact chromoplasts from [1-¹⁴C]acetate was made possible by using ATP, ADP (via adenylate kinase), and, with decreasing efficiency, UTP, CTP, and GTP as energy sources. 2. The glycolytic path from dihydroxyacetone phosphate to acetyl-CoA operates within the chromoplasts. The glycolytic intermediates, especially 2-phosphoglycerate and phosphoenolpyruvate, served as very effective energy donors for fatty acid synthesis by phosphorylating the endogenous adenine nucleotide pool. 3. In the presence of exogenous ATP or ADP, appreciable amounts of in vitro formed fatty acids were found as acyl-CoA and subsequent products, mainly phosphatidylcholine. When other energy sources were used most of the acids formed were in the free form, and to a minor extent, in the phosphatidic acid and diacylglycerol fractions. Similar results have recently been reported for spinach chloroplasts (Kleinig and Liedvogel 1979, FEBS Lett.101, 339–342).Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - UTP uridine triphosphate - CTP cytidine triphosphate - GTP gnanosine triphosphate     

Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent 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 NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km 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.     

Distinct interactions of GTP,UTP, and CTP with G(s) proteins

Early studies showed that in addition to GTP, the pyrimidine nucleotides UTP and CTP support activation of the adenylyl cyclase (AC)-stimulating G(s) protein. The aim of this study was to elucidate the mechanism by which UTP and CTP support G(s) activation. As models, we used S49 wild-type lymphoma cells, representing a physiologically relevant system in which the beta(2)-adrenoreceptor (beta(2)AR) couples to G(s), and Sf9 insect cell membranes expressing beta(2)AR-Galpha(s) fusion proteins. Fusion proteins provide a higher sensitivity for the analysis of beta(2)AR-G(s) coupling than native systems. Nucleoside 5'-triphosphates (NTPs) supported agonist-stimulated AC activity in the two systems and basal AC activity in membranes from cholera toxin-treated S49 cells in the order of efficacy GTP > or = UTP > CTP > ATP (ineffective). NTPs disrupted high affinity agonist binding in beta(2)AR-Galpha(s) in the order of efficacy GTP > UTP > CTP > ATP (ineffective). In contrast, the order of efficacy of NTPs as substrates for nucleoside diphosphokinase, catalyzing the formation of GTP from GDP and NTP was ATP > or = UTP > or = CTP > or = GTP. NTPs inhibited beta(2)AR-Galpha(s)-catalyzed [gamma-(32)P]GTP hydrolysis in the order of potency GTP > UTP > CTP. Molecular dynamics simulations revealed that UTP is accommodated more easily within the binding pocket of Galpha(s) than CTP. Collectively, our data indicate that GTP, UTP, and CTP interact differentially with G(s) proteins and that transphosphorylation of GDP to GTP is not involved in this G protein activation. In certain cell systems, intracellular UTP and CTP concentrations reach approximately 10 nmol/mg of protein and are higher than intracellular GTP concentrations, indicating that G protein activation by UTP and CTP can occur physiologically. G protein activation by UTP and CTP could be of particular importance in pathological conditions such as cholera and Lesch-Nyhan syndrome.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

Regulation of ATP-stimulated releasable myofilaments from cardiac and skeletal muscle myofibrils

The mechanism underlying the formation of easily releasable myofilaments, from myofibrils treated with an ATP-containing relaxing solution, was examined in this investigation. The proportion of releasable myofilaments purified from myofibrils of cardiac, fast- and slow-twitch muscles increased as the [ATP] was raised from 0 to 8.5 mM. The protein composition of the easily releasable myofilaments did not differ with increasing ATP concentrations as observed by 5–15% linear gradient SDS-PAGE. There is a nucleotide specificity to the release of myofilaments in the order of ATP > GTP >> UTP > CTP. Experiments with AMP-PNP and inorganic phosphate (Pi) showed that ATP hydrolysis and the build up of Pi are not requirements in the formation of the easily releasable myofilaments. The release of myofilaments was found to be insensitive to variations in pH from 6.5 to 7.5. The ATP stimulation of myofilament release is ubiquitin-independent, since incubation of purified myofibrils with ubiquitin (1–100 g/ml) at both 20 and 37°C did not change the amount released. Modifying the free sulfhydryl group content by treatment of myofibrils with NEM (0.01–1 mM) or silver nitrate (0.1–10 mM) decreased the proportion of myofilaments that were releasable. Exclusion of 1 mM DTT from the preparation of myofibrils had similar results. These results indicate that the formation of easily releasable myofilaments can be mediated by metabolically related parameters such as the adenosine nucleotides and the reduction-oxidation status of the myofibrillar proteins of striated muscle.     

Effect of nucleoside triphosphate and magnesium ion concentration on the stability and function of rat liver polysomes in vitro

The effects of different concentrations of ATP, GTP, UTP and CTP on polysome stability and function in a cell-free protein-synthesizing system prepared from rat liver were studied. Increasing the concentration of ATP in the incubation medium to 15mm resulted in progressive disaggregation of the polysomes; at ATP concentrations above 2mm their capacity to incorporate amino acids into peptide chains diminished. The same disaggregation phenomenon could be produced by incubating polysomes in a buffered medium containing 5mm-Mg(2+) and increasing concentrations of ATP. Although the disaggregating action of ATP could be prevented by increasing Mg(2+) concentration, the amino acid incorporation in the cell-free protein-synthesizing system remained impaired. The effects of different concentrations of GTP, UTP and CTP on polysome stability were similar to those of ATP. Increasing the concentrations of each nucleoside triphosphate also inhibited the hydrolysis of GTP in the cell-free protein-synthesizing system.     

Coexistence of two ATP sites on the ouabain-complexed (Na+ + K+)-ATPase

When the effects of varying concentrations of ATP on the dissociation rate of the ouabain-enzyme complex were studied, the dissociation rate constant increased with increasing ATP concentrations up to 1 mM, and then decreased with further rise in ATP; indicating that ATP binds to two distinct sites on the complex. ADP and AMP-PNP had similar biphasic effects. GTP, CTP, UTP, and AMP-PCP reduced the dissociation rate. AMP and Pi had no effects. Increase in dissociation rate caused by 0.5 mM ATP was not abolished by saturating CTP, indicating the binding of CTP to only one of the two ATP sites. The data suggest the existence of separate catalytic and regulatory sites, with different affinities and nucleotide specificities.     

The Metabolism of Purine and Pyrimidine Nucleotides in Rat Cortex During Insulin-Induced Hypoglycemia and Recovery

Brains of paralysed rats with insulin-induced hypoglycemia were frozen in situ after spontaneous EEG activity had been absent for 5 or 15 min (“coma”). Recovery (30 min) was achieved in a different group of rats by administering glucose after a 30-min coma period. Purine and pyrimidine nucleotides, nucleosides and free bases were determined in the cortical extracts by high pressure liquid chromatography (HPLC). The ATP values obtained with the HPLC method were in excellent agreement with those obtained using standard enzymatic/fluorometric techniques, while values for ADP and AMP obtained with the HPLC method were significantly lower. Comatose animals showed a severe (40-80%) reduction in the concentrations of all nucleoside triphosphates (ATP. GTP, UTP and CTP) and a simultaneous increase in the concentrations of all nucleoside di- and monophosphates, including that of IMP. The adenine nucleotide pool size decreased to 50% of control level. The concentrations of the nucleosides adenosine, inosine, and uridine increased 50- to 250-fold, while the concentrations of the purine bases, xanthine and hypoxanthine, rose 2- and 30-fold, respectively. There were no increases in the concentrations of adenine, guanine, or xanthosine. Following glucose administration there was a partial (ATP, UTP and CTP) or almost complete (GTP) recovery of the nucleoside triphosphate levels. During recovery, the levels of nucleosidc di- and monophosphates and of adenosine decreased to values close to control; the rise in the inosine level was only partially reversed, and the concentrations of hypoxanthine and xanthine rose further. The adenine nucleotide pool size was only partially restored (to 67% of control value). The adenine nucleotide pool size was not increased by i.p. injection of adenosine or adenine under control condition, or during the posthypoglycemic recovery period.     

Crystal structures of CTP synthetase reveal ATP, UTP, and glutamine binding sites

CTP synthetase (CTPs) catalyzes the last step in CTP biosynthesis, in which ammonia generated at the glutaminase domain reacts with the ATP-phosphorylated UTP at the synthetase domain to give CTP. Glutamine hydrolysis is active in the presence of ATP and UTP and is stimulated by the addition of GTP. We report the crystal structures of Thermus thermophilus HB8 CTPs alone, CTPs with 3SO4(2-), and CTPs with glutamine. The enzyme is folded into a homotetramer with a cross-shaped structure. Based on the binding mode of sulfate anions to the synthetase site, ATP and UTP are computer modeled into CTPs with a geometry favorable for the reaction. Glutamine bound to the glutaminase domain is situated next to the triad of Glu-His-Cys as a catalyst and a water molecule. Structural information provides an insight into the conformational changes associated with the binding of ATP and UTP and the formation of the GTP binding site.     

In situ regulation of mammalian CTP synthetase by allosteric inhibition

The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.