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 class A aspartate transcarbamoylase (ATCase, EC 2.1.3.2) from Pseudomonas fluorescens was purified to homogeneity with retention of full catalytic and regulatory functions. Careful determinations under conditions that minimized proteolysis showed that the molecule is a 1:1 stoichiometric complex of two polypeptide chains of 34 and 45 kDa. Pyridoxal phosphate is a competitive inhibitor of the enzyme (Ki = 1 microM). Reduction of the pyridoxal phosphate enzyme adduct with sodium boro[3H]hydride showed that the active site is located on the 34-kDa polypeptide. Affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an ATP analog, suggested that the regulatory site is also located on the 34-kDa species. While the function of the 45-kDa subunit is unknown, neither carbamoyl phosphate synthetase nor dihydroorotase activities are associated with the ATCase. The molecular mass of the enzyme was determined by gel filtration, sedimentation velocity, and electron microscopy to be 464 kDa. Thus the enzyme is composed of six copies of the 34-kDa polypeptide and six copies of the 45-kDa polypeptide. The molecule has a Stokes' ratio of 70.9 A and a frictional ratio of 1.37, suggesting a compact globular shape. We propose that the P. fluorescens ATCase is composed of two trimers of 34-kDa catalytic chains and is likely to be a D3 dodecamer with an arrangement of subunits analogous to that of the class B ATCase molecules.
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PMID:Subunit structure of a class A aspartate transcarbamoylase from Pseudomonas fluorescens. 823 18

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

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 arg2 gene which encodes the small subunit of carbamoyl phosphate synthetase for Trichoderma virens has been cloned and used to develop a homologous transformation system. A genomic clone containing the arg2 gene was isolated from a cosmid library of T. virens based on complementation of an arginine auxotrophic mutant of this fungus. The predicted amino acid sequence of the arg2 gene shows 56-82% identity with homologous polypeptides from other fungi. It also contains an upstream open reading frame which encodes 24 amino acids. As is observed with other gene sequences encoding this polypeptide in filamentous fungi, the N-terminus of the predicted polypeptide showed characteristic features of a mitochondrial signal sequence. The arg2 gene was used for genetic transformation of T. virens in frequencies of up to 370 transformants/microgram of DNA. Heat-shock treatment of T. virens protoplasts increased the transformation frequency by fivefold, but more than 85% of the transformants were abortive. Both single-copy, homologous integration events and ectopic, non-homologous integration events were detected by Southern analyses of genomic DNA from transformed strains.
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PMID:The arg2 gene of Trichoderma virens: cloning and development of a homologous transformation system. 950 76

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine. The enzyme consists of two polypeptide chains referred to as the large and small subunits. While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia. From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes. Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis. Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E. coli in which His 353 has been replaced with an asparagine residue. Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution. The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.
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PMID:Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis. 963 22

Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5'-pyrophosphate (PRPP) with a dissociation constant of 25 microM. The dissociation constant for UTP of 0.23 mM was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding.
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PMID:Regulation of an Escherichia coli/mammalian chimeric carbamoyl-phosphate synthetase. 981 25

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open beta-sheet surrounded by alpha-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.
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PMID:Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine. 1021 41

The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans. The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid. The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it. Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII. Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway. The 'silent' dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases.
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PMID:Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases. 1041 50

Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli is composed of two polypeptide chains. The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia. Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily. Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP. High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E. coli enzyme are connected by a molecular tunnel of approximately 100 A in total length. Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase. This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit. It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization. In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent. The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site.
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PMID:Carbamoyl-phosphate synthetase. Creation of an escape route for ammonia. 1213 Jun 56


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