Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.5.5 (CPS)
1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Carbamyl phosphate (CP) is synthesized in the liver by two separate enzymes, CPS I and CPS II. CPS I, an intramitochondrial enzyme involved in ureogenesis, has a relative activity of 500- to 1000-fold greater than CPS II, a cytoplasmic enzyme which initiates the sequence of reactions for pyrimidine biosynthesis. The contributions of NH4Cl (substrate for CPS I) ang glutamine (substrate for CPS II) as precursors for pyrimidine biosynthesis in isolated hepatocytes were compared by measuring their effect on uracil nucleotide pool size, the incorporation of NaH14CO3 into these pools, and the accumulation of orotic acid. Physiological concentrations of NH4Cl caused a marked stimulation of incorporation of radioactivity into uracil nucleotides (6-fold increase at 0.5 mM NH4Cl), and radioactive orotate appeared in both the cells and the medium. In contrast, glutamine (at concentrations up to 10 mM) had no effect on the incorporation of radioactivity into uracil nucleotides, and no orotic acid was detected. Uracil nucleotide pools were expanded up to 50% by low levels of NH4Cl, but there was no expansion of this pool in the presence of added glutamine. NH4Cl-driven pyrimidine de novo biosynthesis was insensitive to feedback inhibition by an expanded uracil nucleotide pool, to galactosamine treatment, and to acivicin treatment, indicating that NH+4 stimulated pyrimidine biosynthesis as a result of CP synthesis by mitochondrial CPS I. The consequence of intramitochondrially produced CP being available for pyrimidine biosynthesis is that the controlling step of this pathway (CPS II) is bypassed. The appearance of orotic acid following NH4Cl stimulation indicated that the rate-controlling step of hepatic de novo pyrimidine synthesis under these conditions was orotate phosphoribosyl transferase. These data indicate that, at physiological concentrations of NH+4, the majority of uracil nucleotides synthesized in isolated rat hepatocytes was derived from intramitochondrially generated CP. The effect of NH4Cl on the output of uridine by the isolated perfused rat liver was examined. In the presence of a single addition of 20 mM NH4Cl, the excretion of uridine was increased from 100-200 to 375 nmol h-1 g-1 liver and orotic acid was released into the circulating perfusate reaching a maximum of 2 microM (in 220 ml of perfusate) after 2 h. With 40 mM NH4Cl, uridine export was increased to 450 nmol h-1 g-1 and a maximum of 5 microM orotic acid was released into the perfusate after 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Influence of ammonium ions on hepatic de novo pyrimidine biosynthesis. 298 2

A near full-length cDNA copy of rat carbamoyl-phosphate synthetase I (EC 6.3.4.16) mRNA has been cloned. The cDNA insert in the recombinant plasmid pHN234 is 5.3 kilobases long. Analysis of the sequence coding for carbamoyl-phosphate synthetase I indicates that the gene has arisen from a fusion of two ancestral genes: one homologous to Escherichia coli carA, coding for a glutaminase subunit, and the second homologous to the carB gene that codes for the synthetase subunit. A short amino acid sequence previously proposed to be part of the active site involved in glutamine amide nitrogen transfer in the E. coli and yeast carbamoyl-phosphate synthetases (EC 6.3.5.5) is also present in the rat enzyme. In the mammalian enzyme, however, the glutaminase domain lacks a cysteine residue previously shown to interact with glutamine. The cysteine is replaced by a serine residue. This substitution could, in part, account for the inability of mammalian carbamoyl-phosphate synthetase I to catalyze the hydrolysis of glutamine to glutamic acid and ammonia.
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PMID:The gene coding for carbamoyl-phosphate synthetase I was formed by fusion of an ancestral glutaminase gene and a synthetase gene. 298 6

Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5' proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CP A1 CP A2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH2-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO2H.
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PMID:Nucleotide sequence of the pyrimidine specific carbamoyl phosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene. 303 94

The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO3-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O3, 16O]Pi does not readily participate. These results suggest that the formation of carbamate from MgATP, HCO3-, and glutamine proceeds via a stepwise, not concerted mechanism, involving at least one kinetically competent covalent intermediate, such as carboxyphosphate.
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PMID:Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange. 330 Jul 76

The in vivo actions of two antimetabolites, acivicin (NSC-163501) and tiazofurin (NSC-286193), were examined on the enzymic programs of rat bone marrow. From the bone marrow of the femurs, 100,000 g supernatant fractions were prepared; enzymic activities were measured by isotopic assays, and cellularity was determined. In the normal bone marrow, the specific activities of pyrimidine de novo synthetic enzymes, CDP reductase, dTMP synthase, CTP synthase, carbamoyl-phosphate synthase II (synthase II), orotidine 5'-phosphate decarboxylase and aspartate carbamoyltransferase, were 1, 2.7, 5, 10, 63 and 601 nmol/hr/mg protein, respectively, whereas those of the salvage enzymes, deoxycytidine, thymidine, cytidine and uridine kinases were 3, 43, 149, and 367 nmol/hr/mg protein, respectively. In purine biosynthesis, the activities of the de novo synthetic enzymes, IMP dehydrogenase, formylglycinamidine ribonucleotide (FGAM) synthase, GMP synthase, amidophosphoribosyl-transferase (AT) and adenylosuccinate synthase were 16, 8, 107, 78 and 124 nmol/hr/mg protein, respectively, and those of the salvage enzymes, adenine, hypoxanthine and guanine phosphoribosyl-transferases, were 340, 407, and 1018 nmol/hr/mg protein, respectively. The sequence of events was elucidated after a single i.p. injection of acivicin (5 mg/kg) or tiazofurin (200 mg/kg). Within 2 hr after acivicin injection, CTP, GMP and FGAM synthases lost 85-90%, while AT and synthase II lost 50 and 80%, respectively, of their activities. The activities rose to near normal range by 72-96 hr. The bone marrow cellularity decreased, reaching a nadir at 24 and 48 hr, and returning to normal range by 72 and 92 hr; thymidine kinase activity followed a similar pattern. Tiazofurin injection depressed IMP dehydrogenase activity to 20% by 2 hr with a rebound to normal range by 48 and 72 hr. The cellularity decreased more slowly, reaching its lowest point at 24 hr and returning to normal range at 72 hr. For acivicin the marked depletion of the activities of the glutamine-utilizing enzymes and for tiazofurin that of IMP dehydrogenase might account, in part at least, for the bone marrow toxicity of these antimetabolites. Because of the presence in the bone marrow of high activities of purine and pyrimidine salvage enzymes, it should be possible to design methods utilizing nucleosides and nucleobases to protect the bone marrow from the action of antimetabolites.
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PMID:Enzymic programs of rat bone marrow and the impact of acivicin and tiazofurin. 334

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.
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PMID:Carbamoyl-phosphate synthase in Phycomyces blakesleeanus. 339 22

In Crithidia fasciculata, carbamoyl phosphate synthetase II, which catalyses the first step of de novo pyrimidine biosynthesis, was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. The antitumor drug acivicin competitively inhibited the synthetase II activity with respect to L-glutamine, yielding an apparent Ki of 2 microM. In the absence of L-glutamine, acivicin resulted in a selective, time-dependent inactivation of L-glutamine-dependent activity of the enzyme, with an inactivation constant (Kinact) of 100 microM and a minimum inactivation half-time (T) of 0.2 min. L-Glutamine protected the enzyme from inactivation. These results are consistent with a postulate that acivicin is an active site-directed affinity analogue of L-glutamine, achieving irreversible inactivation. The inactivated enzyme retained ammonia-dependent activity. Acivicin stimulated the ammonia-dependent activity by increasing the Vmax value of the enzyme; apparent Km values for ammonia and MgATP were not affected. Differential action of acivicin on the Crithidia and mammalian synthetase II is discussed.
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PMID:Inactivation of Crithidia fasciculata carbamoyl phosphate synthetase II by the antitumor drug acivicin. 357 57

A high specific activity of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing; EC 6.3.5.5) was demonstrated in extract of the cultured Crithidia fasciculata. The enzyme was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. Apparent Km for the synthetase for L-glutamine, NH4+, MgATP or bicarbonate was 0.27, 26, 1.7 or 1.7 mM at 2.0% dimethyl sulfoxide plus 0.3% glycerol. 8.6% dimethyl sulfoxide plus 1.4% glycerol decreased Km for L-glutamine to 0.10 mM, while Km for MgATP was unaffected. The higher solvent concentrations made Vmax markedly reduced, yielding the inhibition of the activity. These properties are unique to the Crithidia synthetase, compared with the mammalian enzyme.
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PMID:Kinetic properties of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) in the parasitic protozoan Crithidia fasciculata and separation of the enzyme from aspartate carbamoyltransferase. 360 29

Improved methodologies are described which allow the measurement of the part-reactions, with glutamine or ammonia as nitrogen donor, of mammalian carbamoyl-phosphate synthase II (EC 6.3.5.5) through the incorporation of [14C]bicarbonate into either carbamoyl phosphate or carbamoylaspartate. The enzyme is part of the multifunctional polypeptide (CAD) which also comprises the pyrimidine-biosynthetic enzymes aspartate transcarbamoylase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3). The conformational stability of the carbamoyl-phosphate synthase was investigated through the inactivation of the part-reactions which occurred during incubation at 37 degrees C. The domain involved in the removal of the amide N from glutamine was more thermolabile than the ammonia-dependent synthase moiety. The former activity was stabilized in the presence of sodium aspartate or MgATP, whereas the latter was stabilized by MgATP and MgUTP. Binding of MgUTP and MgATP to CAD restricted the initial proteolysis by trypsin and elastase of one or both regions linking the carbamoyl-phosphate synthase domain to the other major domains. A model is described to account for both aspects of nucleotide binding to CAD; these stabilizing effects may be important in the cell, where similar concentrations of nucleotides are found.
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PMID:Nucleotide ligands protect the inter-domain regions of the multifunctional polypeptide CAD against limited proteolysis, and also stabilize the thermolabile part-reactions of the carbamoyl-phosphate synthase II domains within the CAD polypeptide. 363 65

A yeast DNA fragment carrying the gene CP A1 encoding the small subunit of the arginine pathway carbamoyl-phosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CP A1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45 358 and showing extensive homology with the product of carA, the homologous Escherichia coli gene. CP A1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl-phosphate. A comparison of the amino acid sequences of CP A1 polypeptide with the glutamine amidotransferase domains of anthranilate and p-amino-benzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11-residue oligopeptide contains a cysteine which is considered as the active-site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia-dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process. The flanking regions of gene CP A1 have been sequenced, 803 base pairs being determined on the 5' side and 382 on the 3' side. Several features of the 5'-upstream region of CP A1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T-G-A-C-T-C) previously identified in several genes subject to the general control of amino acid biosynthesis.
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PMID:Nucleotide sequence of yeast gene CP A1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Homology of the deduced amino acid sequence to other glutamine amidotransferases. 388 Dec 60


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