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)

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.
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PMID:Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis. 3 10

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

We have measured the 'core' mammalian carbamoyl-phosphate synthetase II (CPSII) activity, using NH4Cl as the nitrogen-donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving Km (NH3) values of 166 microM at high ATP concentrations and 26 microM at low ATP concentrations, while the Km (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the Km (NH3) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The Km for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of activity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5-phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low-affinity UTP-stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.
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PMID:Regulation of the mammalian carbamoyl-phosphate synthetase II by effectors and phosphorylation. Altered affinity for ATP and magnesium ions measured using the ammonia-dependent part reaction. 149 69

On the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes. Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD. In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon. Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge. This bridge connects the subdomains of the CAD CPSase. We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence. The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E. coli CPSase holoenzyme. The fusion in E. coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins.
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PMID:Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion. 151 89

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

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.
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PMID:Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD. 197 79

The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain. 198 78

We have characterized genomic and cDNA clones for arg-2, the gene encoding the small subunit of the Neurospora crassa arginine-specific carbamoyl phosphate synthetase (CPS-A), and examined its transcriptional regulation. The polypeptide's predicted amino acid sequence (453 residues) is 56% and 36% identical with the sequences of the homologous polypeptides of Saccharomyces cerevisiae and Escherichia coli, respectively. The ARG2 polypeptide has an additional amino-terminal domain with the hallmark features of a mitochondrial signal sequence. The arg-2 mRNA also encodes a 24-residue peptide in the segment upstream of the coding region for the ARG2 polypeptide. This upstream open reading frame (uORF) strongly resembles the uORF in the homologous S. cerevisiae transcript. Northern analyses indicate that arg-2 mRNA levels are reduced by arginine supplementation and increased by amino acid limitation. The large increase in arg-2 mRNA levels that occurs in response to amino acid limitation is not observed in a strain containing the cpc-1 mutation, indicating that the cross-pathway control system participates in arg-2 regulation. Four copies of the sequence TGACTC, the binding site for the CPC1 regulatory protein, are found in the arg-2 genetic region. Two copies are located upstream of the mRNA start sites, and two are present within introns in the arg-2 uORF.
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PMID:The Neurospora crassa arg-2 locus. Structure and expression of the gene encoding the small subunit of arginine-specific carbamoyl phosphate synthetase. 214 6

The arcABC operon of Pseudomonas aeruginosa encodes arginine deiminase, catabolic ornithine carbamoyltransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and arcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N-terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175-180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mg2+ or Mn2+ and strongly inhibited by Zn2+. The apparent Km for L-arginine was 0.04 mM in the presence of Mg2+ and 0.47 mM without Mg2+. The arcC open reading frame codes for a 33-kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation-initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression. Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C-terminal part of carbamoyl-phosphate synthetase (large subunit) from Escherichia coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. coli.
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PMID:Sequence analysis and expression of the arginine-deiminase and carbamate-kinase genes of Pseudomonas aeruginosa. 253 2

Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.
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PMID:Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain. 254 74


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