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)

Total RNA or poly(A)(+) RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.
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PMID:Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver. 22 76

Biotin carboxylase [biotin-carboxyl-carrier-protein:carbon-dioxide ligase (ADP-forming), EC 6.3.4.14] is the enzyme mediating the first step of the acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] reaction. We screened an Escherichia coli DNA library and a DNA fragment carrying the biotin carboxylase gene fabG, and its flanking regions were cloned. The gene for biotin carboxyl carrier protein was found 13 base pairs upstream of the fabG gene. Nucleotide sequencing of the recombinant plasmids revealed that the fabG codes for a 449-amino acid residue protein with a calculated molecular weight of 49,320, a value in good agreement with that of 51,000 determined by SDS/polyacrylamide gel electrophoresis of the purified enzyme. The deduced amino acid sequence of biotin carboxylase is also consistent with the partial amino acid sequence determined by Edman degradation. The primary structure of this enzyme exhibits a high homology with those of other biotin-dependent enzymes and carbamoyl-phosphate synthetase [carbon-dioxide:L-glutamine amino-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]; therefore, all these enzymes probably function through the same mechanism of reaction.
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PMID:Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit. 168 20

Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.
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PMID:Mammalian dihydroorotase: nucleotide sequence, peptide sequences, and evolution of the dihydroorotase domain of the multifunctional protein CAD. 196 94

The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver and dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. Synthesis of citrulline by isolated mitochondria from ornithine proceeds at a near optimal rate at ornithine concentrations as low as 0.08 mM. The same stoichiometry and rates of citrulline synthesis are observed when ornithine is replaced by arginine. The mitochondrial location of arginase does not appear to reflect a mechanism for regulating ornithine availability.
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PMID:Submitochondrial localization of arginase and other enzymes associated with urea synthesis and nitrogen metabolism, in liver of Squalus acanthias. 286 47

The large subunit of carbamoyl phosphate synthase A [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] from Neurospora crassa is encoded by a nuclear gene but is localized in the mitochondrial matrix. We have utilized N. crassa strains that produce both normal and carboxyl-terminal-truncated forms of carbamoyl phosphate synthase A to ask whether the carboxyl terminus affects import of the carbamoyl phosphate synthase A precursor. We found that carboxyl-terminal-truncated precursors were directed to mitochondria but that they were imported less efficiently than full-length proteins that were synthesized in the same cytoplasm. Our results suggest that effective import of proteins into mitochondria requires appropriate combinations of targeting sequences and three-dimensional structure.
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PMID:Carboxyl-terminal sequences influence the import of mitochondrial protein precursors in vivo. 295 46

Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl2, NH4Cl, and the activator N-acetyl-L-glutamate. These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species and a 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH4Cl and N-acetyl-L-glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.
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PMID:Controlled proteolysis of ammonia-dependent carbamoyl-phosphate synthetase I from Syrian hamster liver. 325 64

The pyr-3 gene of Neurospora crassa codes for the bifunctional enzyme pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase (carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating)/carbamoylphosphate: L-aspartate carbamoyltransferase), EC 6.3.4.16/EC 2.1.3.2). We describe the investigation of substrate- and product-binding sites of the enzyme by affinity chromatography, using the ligands aspartate, glutamate, and adenosine 5'-diphosphate, and investigate the channelling of carbamoyl phosphate, the product of the first function and substrate of the second, through the pathway. For this latter aspect of the investigation, two new enzyme assays were devised and described. The results of the competition studies on carbamoyl phosphate-binding are consistent with the existence of two different binding sites within the enzyme for this metabolic intermediate, one for it as the product of the first step and the other for it as the substrate of the second.
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PMID:Investigation of binding sites in the complex pyrimidine-specific carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa. 621 40

Several recombinant plasmids containing cpaII, the gene that encodes the large subunit of yeast arginine-specific carbamoyl-phosphate synthetase [carbamoyl-phosphate synthetase (glutamine-hydrolyzing), carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.3.5], have been isolated. The plasmids were selected by transformation of a yeast strain with a mutation in the structural gene of the large subunit of carbamoyl-phosphate synthetase. By using a recombinant pool with inserts of yeast nuclear DNA of 5-20 kilobase pairs, we obtained 13 transformants. Of five transformants studied, three have been found to have stable plasmid inserts. These plasmids could be amplified in Escherichia coli and transferred back into the yeast carbamoyl-phosphate synthetase-deficient strains with concomitant complementation of the nuclear mutation. Plasmids pJL2/T1 and pJL2/T5 contain identical nuclear DNA inserts of 5.9 kilobase pairs. Although the insert of plasmid pJL2/T3 is also 5.9 kilobase pairs long, the sequence overlap with pJL2/T1 and pJL2/T5 is only 4.5 kilobase pairs long. The T3 insert has an orientation in the vector opposite to that of the T1 and T5 inserts. The recombinant plasmids with the yeast cpaII gene fail to cross-hybridize with a cloned fragment of E. coli DNA containing the carA and carB genes for the bacterial carbamoyl-phosphate synthetase.
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PMID:Cloning of a yeast gene coding for arginine-specific carbamoyl-phosphate synthetase. 628 75

The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.
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PMID:DNA sequence of the carA gene and the control region of carAB: tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12. 633 Jul 44

The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650,000 to 100,000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultraviolet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibtors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.
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PMID:The involvement of proteolysis in conformational stability of the carbamoyl-phosphate synthetase/aspartate carbamoyltransferase enzyme of Neurospora crassa. 645 44


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