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)

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.
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PMID:Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish). 257 May 70

Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide.
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PMID:Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain. 274 25

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics. 287 Oct 22

Acetyl-coenzyme A carboxylase (Ac-CoA carboxylase; EC 6.4.1.2) catalyzes the rate-limiting reaction in long-chain fatty acid biosynthesis. To investigate the mechanism of genetic control of expression of Ac-CoA carboxylase and the relationship between its structure and function, cDNA clones for Ac-CoA carboxylase were isolated. The complete coding sequence contains 7035 bases; it encodes a polypeptide chain of 2345 amino acids having a Mr of 265,220. The sequences of several CNBr peptides of Ac-CoA carboxylase were localized within the predicted protein sequence as were those peptides that contain the sites for phosphorylation. The deduced protein contains one putative site for biotinylation in the NH2-terminal half. The "conserved" biotinylation site peptide, Met-Lys-Met, is preceded by valine, whereas alanine is found in a similar position in all other known biotin-containing proteins. The primary sequences of Ac-CoA carboxylase and carbamoyl phosphate synthetase exhibit substantial identity.
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PMID:Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase. 290 Oct 88

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

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

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

An in vitro system reconstituted with mouse liver polysome translation products was used to study the nature of polypeptide species imported into mitochondria from different mouse tissues such as liver, kidney, brain, and heart, as well as from Ehrlich ascites, Novikoff hepatoma, and Morris hepatoma 3924A tumor lines. Mouse hepatic mitochondria import a number of proteins including 160-kilodalton (kDa) carbamoyl-phosphate synthetase I (CPS-I). Two other proteins of 63 and 57 kDa of unknown function are also imported as major components by mouse liver mitochondria. Under these in vitro conditions, however, mitochondria from non-CPS-I expressing tissues such as brain, kidney, and heart failed to import and process the precursor forms of CPS-I (pCPS-I). Furthermore, mitochondria from three different tumor lines (Novikoff hepatoma, Morris hepatoma, and Ehrlich ascites) containing negligible CPS-I activity were also unable to import and process pCPS-I to any significant level. Similarly, the 63-kDa protein was selectively transported into liver and kidney mitochondria and also into Ehrlich ascites mitochondria at reduced levels, but not into mitochondria from heart and brain. Nevertheless, the 57-kDa protein and a number of proteins of less than 45 kDa are transported efficiently by all of the mitochondrial types studied. These results provide evidence for tissue- or cell-specific selectivity at the mitochondrial membrane level for the transport of some proteins. The transports of 63- and 57-kDa proteins are differentially inhibited by mouse liver mitochondrial matrix and membrane fractions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transport of proteins into hepatic and nonhepatic mitochondria: specificity of uptake and processing of precursor forms of carbamoyl-phosphate synthetase I. 409 60

Carbamyl phosphate synthetase (from Escherichia coli) consists of a 7.3S protomeric unit that contains one heavy polypeptide chain (molecular weight about 130,000) and one light chain (molecular weight about 42,000). The heavy and light chains were separated by gel filtration in the presence of 1 M potassium thiocyanate. In contrast to the native enzyme and the reconstituted enzyme (prepared by mixing the separated heavy and light chains), the heavy chain does not catalyze glutamine-dependent carbamyl phosphate synthesis, although it does catalyze the synthesis of carbamyl phosphate from ammonia. The heavy chain also catalyzes two of the partial reactions catalyzed by the intact enzyme; i.e., the bicarbonate-dependent cleavage of ATP and the synthesis of ATP from ADP and carbamyl phosphate. Both positive (ammonia, ornithine, IMP) and negative (UMP) allosteric regulatory sites are located on the heavy chain. The only catalytic activity exhibited by the light chain is the hydrolysis of glutamine. A model is presented according to which glutamine binds to the light chain, which is followed by release of nitrogen from the amide group for use by the heavy chain. The findings suggest that glutamine-dependent carbamyl phosphate synthetase (and perhaps other glutamine amidotransferases) arose in the course of evolution by a combination of a primitive ammonia-dependent synthetic enzyme and a glutaminase; this combination may have been associated with a change from ammonia to glutamine as the principal source of nitrogen.
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PMID:Reversible dissociation of carbamyl phosphate synthetase into a regulated synthesis subunit and a subunit required for glutamine utilization. 494 34


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