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)

Two carbamyl phosphate synthetases, the first an arginine-synthetic enzyme (CPS(arg)) and the second a pyrimidine-synthetic enzyme (CPS(pyr)), are shown to be present in Neurospora. The two enzymes can be separated on the basis of size and are distinguished by several different properties. Both CPS(pyr) and CPS(arg) have substrate requirements of adenosine triphosphate, HCO(3) (-), and l-glutamine, although NH(4) (+) in high concentration will partially replace glutamine. CPS(pyr) activity can be completely inhibited by 5 x 10(-4) to 10 x 10(-4)m uridine triphosphate (UTP). CPS(pyr) is cold-labile and can be protected against cold inactivation by UTP. The synthesis of CPS(pyr) and aspartate transcarbamylase (ATC), the initial enzymatic steps of the pyrimidine pathway, are co-derepressed by pyrimidine starvation. Mutations affecting CPS(pyr) and ATC all map at the same locus, pyr-3. Three classes of mutants with respect to the two activities were found: CPS(+)ATC(-), CPS(-)ATC(+), and CPS(-)ATC(-). The distribution of these mutants on the genetic map, together with other data, indicate that the two activities are carried by a bifunctional protein.
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PMID:Pyrimidine-specific carbamyl phosphate synthetase in Neurospora crassa. 543 4

Biochemical steps of the pyrimidine pathway have been found to be the same in yeast as in bacteria, and all except one step have been characterized. The activities of the first two enzymes, carbamoyl phosphate synthetase and aspartic transcarbamylase, are simultaneously controlled by feedback inhibition and repression. Moreover, these enzymes are coded by the same genetic region (ura-2) and seem to form a single enzymatic complex. The enzymes that follow later in the pathway are induced in a sequential way by the intermediary products and are insensitive to pyrimidine repression. The corresponding genes (ura-4, ura-1, ura-3) are not linked to each other or to ura-2, the gene for carbamoyl phosphate synthetase and aspartic transcarbamylase. Mutants that have simultaneously lost feedback inhibition by uridine triphosphate for carbamoyl phosphate synthetase and for aspartic transcarbamylase have been found and mapped in the gene ura-2.
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PMID:Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. 565 25

Glutamine-dependent carbamoyl-phosphate synthetase, the first enzyme of the de novo biosynthetic pathway for pyrimidine nucleotides, was purified about twenty-fold from 105 000 x g supernatant of the Ascaris ovary homogenate. The enzyme activity was feedback-inhibited by UDP and UTP while it was stimulated by 5-phosphoribosyl 1-pyrophosphate. Most of the catalytic and regulatory properties of the Ascaris synthetase were similar to those of the mammalian synthetase. A significant difference is that the Ascaris enzyme was more strongly inhibited by UDP than by UTP whereas the mammalian enzyme is more sensitive to UTP than to UDP. The Ascaris enzyme was also inhibited by other various nucleoside diphosphates, such as dUDP, dADP and CDP, generally more strongly than by the corresponding nucleoside triphosphates. Aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway, were also demonstrated in the supernatant fraction. These two enzymes were copurified with the synthetase and the relative activities of the three enzymes remained nearly constant (1:850-890:50-60) throughout the purification. In a sucrose gradient centrifugation, the enzymes cosedimented as a single peak with a sedimentation coefficient (s20,w) of about 32 S under the condition used. These results strongly suggest that the enzymes exist as a multienzyme complex similar to those found in higher animals. The activity of the carbamoyltransferase was insensitive to nucleotides and related compounds. These results indicate that the synthetase plays a key role in the control of pyrimidine biosynthesis in the Ascaris ovary.
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PMID:Control of pyrimidine biosynthesis in the Ascaris ovary: regulatory properties of glutamine-dependent carbamoyl-phosphate synthetase and copurification of the enzyme with aspartate carbamoyltransferase and dihydroorotase. 610 8

Carbamoyl-phosphate synthetase II [EC 6.3.5.5] of rat ascites hepatoma cells (AH 13), the first and regulatory enzyme of de novo pyrimidine nucleotide biosynthesis, exists as a multienzyme complex (molecular weight, 870,000) with aspartate carbamoyltransferase [EC 2.1.3.2] and dihydroorotase [EC 3.5.2.3] (Mori, M. & Tatibana, M. (1975) J. Biochem. 78, 239-242). The purified complex was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase [EC 2.7.1.37] of rabbit skeletal muscle. The incorporation of 32Pi was 2.2 mol/mol of the complex. The phosphorylation was completely inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with y inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with y inhibited by the inhibitor protein of the cAMP-dependent protein kinase. Among the substrates and effectors of the enzyme complex tested, only MgUTP, an allosteric inhibitor of carbamoyl-phosphate synthetase II, strongly inhibited the phosphorylation; this inhibition was due probably to the competition of MgUTP with the substrate MgATP for the protein kinase. The complex that was phosphorylated by cAMP-dependent protein kinase was dephosphorylated by phosphoprotein phosphatase [EC 3.1.3.16] of rat skeletal muscle. The complex was also phosphorylated by cAMP-independent protein kinase activity present in the extract of AH 13 cells and dephosphorylated by phosphoprotein phosphatase activity of the same origin. These results suggest that the complex is subject to phosphorylation and dephosphorylation in the living cells. Phosphorylation of the complex by cAMP-dependent protein kinase was associated only with a slight change, albeit definite, in the activity of carbamoyl-phosphate synthetase II under the assay conditions. Thus, the physiological significance of phosphorylation-dephosphorylation remains to be further studied.
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PMID:Phosphorylation and dephosphorylation of carbamoyl-phosphate synthetase II complex of rat ascites hepatoma cells. 611 55

The multifunctional protein that initiates de novo pyrimidine biosynthesis in mammalian cells carries carbamoylphosphate synthetase, aspartate transcarbamylase (aspartate carbamoyltransferase), and dihydro-orotase activities on a single 215,000-dalton polypeptide chain. Kinetic studies of the controlled proteolysis of the molecule by elastase showed that the protein was not attacked at random by the protease but rather was successively cleaved into at least six well-defined proteolytic fragments. The initial cleavage converted the intact molecule into a 190,000-dalton species which appeared to retain all of the catalytic and regulatory functions of the native protein. This species was subsequently cleaved into two fragments, 150,000 and 40,000 daltons. The 40,000-dalton species, which carried the aspartate transcarbamylase activity, was resistant to further proteolysis; the 150,000-dalton polypeptide, which carried carbamoyl-phosphate synthetase and dihydro-orotase activities, underwent further digestion to 140,000 daltons. Continued proteolysis produced two species, 79,000 and 45,000 daltons; like the 40,000-dalton species, these were stable against further elastase digestion. The aspartate transcarbamylase and dihydro-orotase activities and the regulatory functions were preserved throughout the course of digestion; the carbamoylphosphate synthetase activity was more labile. By using sucrose gradient centrifugation and ion exchange chromatography, the 40,000- and 45,000-dalton species have been isolated. The 40,000-dalton fragment was found to have only aspartate transcarbamylase activity; the 45,000-dalton fragment has only dihydro-orotase activity. These experiments showed that this multifunctional protein is organized as discrete structural domains in which regions of the polypeptide chain are autonomously folded into separate functional units.
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PMID:Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells: evidence for discrete structural domains. 611 66

Carbamoyl-phosphate synthetase II of higher animals, the first enzyme of de novo pyrimidine biosynthesis, forms a multienzyme complex with aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway. The hypothesis that the complex serves to channel carbamoyl-phosphate, synthesized by the first enzyme of the complex, to the second enzyme was tested using a highly purified complex preparation from Yoshida ascites hepatoma cells (AH 13). Experimentally, aspartate carbamoyltransferase in the complex was allowed to compete with exogenously added ornithine carbamoyltransferase, another carbamoyl-phosphate-utilizing enzyme, for carbamoyl-phosphate which was either synthesized endogenously or added exogenously. The ratios of amounts of the two enzymic products, carbamoyl-aspartate and citrulline, were compared. In the absence of enzyme stabilizers dimethyl sulfoxide or glycerol, a slight channeling of the intermediate in the complex was observed. The further addition of 5-phosphoribosyl 1-pyrophosphate, MgUTP (positive and negative allosteric effectors of carbamoyl-phosphate synthetase II), 30% (v/v) dimethyl sulfoxide or 30% (w/v) glycerol did not affect the extent of channeling. It was slightly increased in the presence of 7.5% (v/v) dimethyl sulfoxide plus 2.5% (w/v) glycerol. Any shift of the assay temperature, pH or concentration of MgATP or of the enzyme complex resulted in little further increase in the extent of channeling. Even when a larger amount of the enzyme complex was used to approximate physiological conditions, there was no increase in the extent of channeling either without or with allosteric effectors. MgUTP even abolished channeling under these conditions. These results indicate that carbamoyl-phosphate can be channeled in the multienzyme complex of AH 13 cells, but the extent of channeling is very small, contrary to expectation.
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PMID:Studies on channeling of carbamoyl-phosphate in the multienzyme complex that initiates pyrimidine biosynthesis in rat ascites hepatoma cells. 613 83

The first three enzymes of pyrimidine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyl-transferase, and dihydro-orotase) are carried on a multifunctional protein in mammalian cells and are on separate proteins in bacteria. A plasmid containing a cDNA sequence corresponding to 80% of a hamster mRNA for this protein was transformed into Escherichia coli mutants lacking aspartate carbamoyltransferase (pyrB) or dihydro-orotase (pyrC). Only pyrB transformants were able to grow in the absence of uracil. Plasmid recovered from primary transformants was similar in size to the original plasmid and could yield prototrophs after secondary transformation of E. coli pyrB mutants. When cell extracts were prepared from pyrB transformants, high levels of aspartate carbamoyltransferase activity were found, and the enzyme had properties identical to the mammalian enzyme, including lack of allosteric regulation, precipitation by antiserum specific to the hamster multifunctional protein, and presence of a strong aggregation center. These results demonstrate that (i) a partial hamster protein can complement E. coli defective in pyrimidine biosynthesis, (ii) the order of the enzyme domains of the multifunctional protein is likely to be NH2-dihydro-orotase-carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-COOH, and (iii) the enzyme domains appear to be self-contained at the DNA and protein levels. The protocol described here may be a general means for studying the domains of multifunctional proteins and for isolating other mammalian genes for which bacterial mutants have been prepared. It also permits study of the structure and function of the same gene in both prokaryotic and eukaryotic cells and may provide new insight into the evolution of complex genes.
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PMID:Partial cDNA sequence to a hamster gene corrects defect in Escherichia coli pyrB mutant. 613 12

The 6 enzymes involved in de novo synthesis of pyrimidines were measured in Plasmodium falciparum isolated by saponin lysis from RBC's nonsynchronized and synchronized in vitro cultures. The total activities were found to be dependent on the stage of the P. falciparum cycle. In parasites isolated from synchronized cultures, the highest activities for all enzymes were found at about 27 hr after synchronization in the late trophozoite stage, or just before schizont formation. Merozoites and ring forms contained little de novo activity. The first enzyme of the pathway, carbamyl phosphate synthetase (CPS-II) preferentially utilized glutamine. Ammonia was a poor substrate. CPS-II was unstable in the absence of the cryoprotectants, dimethylsulfoxide and glycerol. The apparent Km for MgATP--was 3.8 +/- 0.7 mM and the enzyme in all morphological forms of P. falciparum (ring, mature trophozoites and schizonts) was inhibited by UTP. The activity of the fourth enzyme of the pathway, dihydroorotate dehydrogenase, appeared to be linked to the cell's respiratory chain; inhibitors of mammalian electron transport such as cyanide, amytal, antimycin A, thenoyltrifluoroacetone and ubiquinone analogs also inhibited the P. falciparum enzyme. The demonstration of the variation of activity of the pyrimidine enzymes correlates with the increased synthesis of nucleic acids in the late trophozoite stage. These observations provide a basis for the testing of the effectiveness of pyrimidine analogs as potential antimetabolites against various forms of the parasite.
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PMID:Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum. 615 76

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.
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PMID:Multi-enzyme-targeted chemotherapy by acivicin and actinomycin. 618 Jun 9

Brief exposure to 6-azauridine stimulates the production of carbamoyl phosphate for de novo pyrimidine biosynthesis in vitro in slices of haematopoietic spleen from anaemic mice (preceding paper). In studies of the underlying mechanism for this response we turned our attention to changes in the level of substrates and effectors for carbamoyl-phosphate synthetase II. Intermediates of the orotic acid pathway and 6-azauridine had little effect on the synthetase activity in vitro. 6-Azauridine 5'-monophosphate (6-AzaUMP) stimulated synthetase II, possibly in an allosteric manner. However, in view of the potency as an activator and the tissue levels, 6-azaUMP may be only partially responsible for the stimulation. Adenine nucleotide levels in the tissue showed only minor changes after brief exposure (15 min) to 6-azauridine. The level of UTP and UDP, potent inhibitors for synthetase II, showed no significant change. The level of 5-phosphoribosyl 1-pyrophosphate (PPRibP), a potent positive effector for the synthetase II, showed a more than 1.5-fold increase after 15 min. The relative importance of these factors was evaluated by assay of the synthetase, partially purified from mouse spleen, under simulated conditions in vitro. The results indicated that the enhanced level of PPRibP played a major role in increasing the production of carbamoyl phosphate. In Ehrlich ascites cells in vitro, where 6-azauridine did not increase carbamoyl phosphate production, the basal PPRibP level was high (range over 0.1 mM) and the changes in the level, brought about by the analogue, were relatively small.
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PMID:Enhancement of intracellular 5-phosphoribosyl 1-pyrophosphate levels as a major factor in the 6-azauridine-induced stimulation of carbamoyl phosphate synthesis in mouse spleen slices. 618 35


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