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)

Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9-36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene.
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PMID:Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments. 302 1

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.
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PMID:Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. 334 46

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

Most DNA replication origins in eukaryotes localize to nontranscribed regions, and there are no reports of origins within constitutively expressed genes. This observation has led to the proposal that there may be an incompatibility between origin function and location within a ubiquitously expressed gene. The biochemical and functional evidence presented here demonstrates that an origin of bidirectional replication (OBR) resides within the constitutively expressed housekeeping gene CAD, which encodes the first three reactions of de novo uridine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase). The OBR was localized to a 5-kb region near the center of the Syrian hamster CAD transcriptional unit. DNA replication initiates within this region in the single-copy CAD gene in Syrian baby hamster kidney cells and in the large chromosomal amplicons that were generated after selection with N-phosphonacetyl-L-aspartate, a specific inhibitor of CAD. DNA synthesis also initiates within this OBR in autonomously replicating extrachromosomal amplicons (CAD episomes) located in an N-phosphonacetyl-L-aspartate-resistant clone (5P20) of CHOK1 cells. CAD episomes consist entirely of a multimer of Syrian hamster CAD cosmid sequences (cCAD1). These data limit the functional unit of replication initiation and timing control to the 42 kb of Syrian hamster sequences contained in cCAD1. In addition, the data indicate that the origin recognition machinery is conserved across species, since the same OBR region functions in both Syrian and Chinese hamster cells. Importantly, while cCAD1 exhibits characteristics of a complete replicon, we have not detected autonomous replication directly following transfection. Since the CAD episome was generated after excision of chromosomally integrated transfected cCAD1 sequences, we propose that prior localization within a chromosome may be necessary to "license" some biochemically defined OBRs to render them functional.
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PMID:Identification of an origin of bidirectional DNA replication in the ubiquitously expressed mammalian CAD gene. 762 8

We have demonstrated biochemically that the conformation of the proteolytic fragment (mammalian aspartate transcarbamoylase) from the C-terminus of the 240-kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross-reacts with antibodies raised against the E. coli enzyme. CAD is digested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195-kDa 'nicked CAD' molecule containing active carbamoyl phosphate synthetase. These two products are easily separated by ion-exchange chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N-terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion-exchange columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.
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PMID:Proteolytic cleavage of the multienzyme polypeptide CAD to release the mammalian aspartate transcarbamoylase. Biochemical comparison with the homologous Escherichia coli catalytic subunit. 795 21

1. Carbamoyl-phosphate synthetase (EC 6.3.5.5.) catalyses the synthesis of carbamoyl phosphate, the immediate precursor of arginine and pyrimidine biosynthesis, and is highly conserved throughout evolution. The large subunit of all carbamoyl-phosphate synthetases sequenced to date comprises two highly homologous halves, the product of a proposed ancestral gene duplication. The sequences of the enzymes of Escherichia coli, Drosophila melanogaster, Saccharomyces cerevisiae, rat and Syrian hamster all have duplications, suggesting that this event occurred in the progenote predating the separation of the major phylae. Until now, only limited data on carbamoyl-phosphate synthetase were available for the primitive eukaryote Dictyostelium discoideum and for the Archaea Methanosarcina barkeri MS. The DNA sequence of the D. discoideum carbamoyl-phosphate gene and additional sequence for the carbamoyl-phosphate synthetase gene of M. barkeri MS have been determined, and a duplicated structure for both is clearly demonstrated. 2. Genes with ancient duplications provide unique information on their evolution. A study of the intron/exon organization of the rat carbamoyl-phosphate synthetase I gene and the carbamoyl-phosphate synthetase hamster II gene in the CAD multi-gene complex shows that at least some of their introns are very old. Evidence is provided that some introns must have been present in the ancestral precursor before its duplication. 3. The human carbamoyl-phosphate synthetase I gene has been isolated and characterized. A human liver cDNA library was constructed and probed for carbamoyl-phosphate synthetase I. A human genomic DNA cosmid library was also probed for the carbamoyl-phosphate synthetase I gene. The cDNA sequence of the human carbamoyl-phosphate synthetase I gene has been determined, and work has been initiated to confirm that at least part of this gene is contained within two cosmids spanning 46kb. This will enable future studies to be made on mutations in this gene in the rare autosomal recessive deficiency of carbamoyl-phosphate synthetase I.
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PMID:Molecular studies on an ancient gene encoding for carbamoyl-phosphate synthetase. 838 76

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.
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PMID:Activation by fusion of the glutaminase and synthetase subunits of Escherichia coli carbamyl-phosphate synthetase. 924 57

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5'-phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell. We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.
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PMID:A reciprocal allosteric mechanism for efficient transfer of labile intermediates between active sites in CAD, the mammalian pyrimidine-biosynthetic multienzyme polypeptide. 928 32

The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.
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PMID:The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain. 985 83

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.
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PMID:A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila. 1008 Aug 91


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