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)

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

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

The first reaction in pyrimidine and arginine biosynthesis in Escherichia coli is catalyzed by a single enzyme, carbamoyl-phosphate synthetase (EC 6.3.5.5), the product of the carAB operon. Expression of this operon is cumulatively repressed by arginine and pyrimidines. The nucleotide sequence of the carAB control region was determined and transcriptional starts were localized. Two adjacent promoters, 70 base pairs apart, appear to be used in vivo, the downstream one overlapping a typical arginine operator. The absence of any attenuation-like sequence excludes such a mechanism for pyrimidine-mediated repression. Various fragments of the carA promoter-proximal region were fused in vitro with the lacZ gene. Results obtained with these fusions indicate that (i) translation of the carA gene can be initiated in vivo without an AUG codon but very likely with an UUG or an AUU codon; (ii) the carAB downstream promoter is repressed by arginine; and (iii) the carAB upstream promoter is repressed by pyrimidines and subject to stringent control. When carried by a multicopy plasmid the carAB control region escapes repression by arginine and pyrimidines. The existence of a pyrimidine repressor, present in limiting amounts in the cell, is therefore postulated.
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PMID:Multiple regulatory signals in the control region of the Escherichia coli carAB operon. 637 9

Aspartate transcarbamoylase (ATCase) is purified from wheat germ as a monofunctional trimer of 36 kDa chains. The possibility that this may be a proteolytic fragment of a large endogenous complex in which ATCase is covalently fused to other pyrimidine-pathway enzymes (such as exists in animals or fungi) was tested. Examination of a rabbit antiserum raised against the purified enzyme confirmed the presence of anti-(wheat ATCase) antibodies by several independent methods. Extracts of wheat seedlings prepared under non-proteolysing conditions were challenged by the antiserum, and in some cases by purified anti-(36 kDa ATCase) antibodies, using immunoblotting techniques. The 36 kDa species was the dominant immunopositive polypeptide. However, the extract also contained small amounts of two larger (45 and 55 kDa) immunopositive polypeptides, as well as traces of polypeptides smaller than 36 kDa, which were assumed to be minor proteolytic products. Neither of the 45 or 55 kDa polypeptides is large enough to also incorporate a carbamoyl phosphate synthetase or dihydroorotase of the sizes found in other organisms. They may be targeted precursors of ATCase with intact leader sequences. A screen of a wheat cDNA expression library by the anti-(ATCase) serum yielded a single positive clone which was shown, by DNA sequencing, to be a concatemer of two cDNAs, one of which encoded a partial ATCase. Northern analysis using this clone as probe identified two transcripts of about 1.3 and 1.0 kbp, but showed no evidence of a transcript of 2 kbp or greater which would be required to encode a bifunctional polypeptide. These results confirm that wheat ATCase is not translationally fused to dihydroorotase or carbamoylphosphate synthetase, as it is in animals and fungi. The deduced amino-acid sequence of the partial wheat ATCase is compared with the catalytic chain of the ATCase from Escherichia coli and with other ATCases.
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PMID:Endogenous polypeptide-chain length and partial sequence of aspartate transcarbamoylase from wheat, characterised by immunochemical and cDNA methods. 807 53

In the protozoan parasite, Babesia bovis, the glutamine-dependent carbamoyl phosphate synthetase-encoding gene (CPSII) contains contiguous amidotransferase- and synthetase-encoding sequences. Unlike the organisation in most eukaryotes, the gene is not fused with other genes encoding enzymes of pyrimidine biosynthesis de novo. The nucleotide sequences immediately upstream and downstream from the gene contain motifs which may be involved in regulating its expression.
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PMID:Sequences upstream and downstream from the glutamine-dependent carbamoyl phosphate synthetase-encoding gene from the protozoan Babesia bovis. 865 85

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.
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PMID:Function of the major synthetase subdomains of carbamyl-phosphate synthetase. 866 13

The amidotransferase or glutaminase domain (GLN domain) of mammalian carbamyl-phosphate synthetase II (CPSase II) catalyzes glutamine hydrolysis and transfers ammonia to the synthetase domain (CPS domain), where carbamyl phosphate formation is catalyzed in three consecutive reactions. The GLN and CPS domains are part of a single polypeptide and are connected via a 29-amino acid chain segment (GC linker). In contrast, the two comparable domains of Escherichia coli CPSase are not fused, but are separate, noncovalently associated subunits. To establish the function of the GC linker in mammalian CPSase, it was deleted, and the two domains were directly fused. The deletion mutant not only catalyzed glutamine-dependent carbamyl phosphate synthesis, but was activated 10-fold relative to its wild-type counterpart. However, ammonia-dependent synthesis of carbamyl phosphate was abolished, indicating that ammonia no longer had access to the active site on the CPS domain. The mutant was still sensitive to inhibition by the allosteric effector UTP, but was no longer activated by the allosteric effector phosphoribosyl pyrophosphate, although evidence indicated that the latter could bind to the enzyme. The linker appears to serve as a spacer that allows the complex to cycle between two conformations, an open low activity form in which the ammonia site on the CPS domain is accessible and an activated conformation in which the ammonia generated in situ from glutamine is directly channeled to the CPS active site and access to exogenous ammonia is blocked.
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PMID:Trapping an activated conformation of mammalian carbamyl-phosphate synthetase. 924 56

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

The first two steps of the de novo pyrimidine biosynthetic pathway in Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously produced carbamoyl phosphate is sequestered and channeled to the ATCase active site.
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PMID:Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain. 1044 40


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