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)

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

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis. 764 1

The reactive cysteine residue within the small subunit of Escherichia coli carbamoyl phosphate synthetase has been identified using the technique of site-directed mutagenesis. Three cysteine residues have previously been found to react with N-ethylmaleimide (NEM) under controlled reaction conditions. Two of these cysteine residues are found on the large subunit, while the third cysteine is located on the small subunit. In the present investigation, Cys-248 of the small subunit has been identified as the residue that reacts with NEM in the presence of MgATP and bicarbonate. Three cysteine residues of the small subunit at positions 131, 214, and 248 were individually mutated to serine residues. These site-specific changes, in addition to N-ethylmaleimide-labeling studies, demonstrated that Cys-248 is the amino acid that reacts with N-ethylmaleimide. Substitution of Cys-248 of the small subunit with larger residues (Asp, Phe, Arg, and Trp) was conducted in order to more closely mimic the observed properties of the NEM-labeled enzyme. The partial glutaminase activity of the C248D mutant increased 40-fold relative to the wild-type enzyme, while the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Similar, but less dramatic, effects were observed for the other mutants, C248S, C248R, C248F, and C248W. There was good correlation between the extent of enhancement of the partial glutaminase activity and an uncoupling of the phosphorylation reactions that occur on the large subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A molecular wedge for triggering the amidotransferase activity of carbamoyl phosphate synthetase. 813 Feb 8

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 carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS. B from E. coli and mammalian CPSase have shown that homologous dimers of either of these "half-molecules" could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14-16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.
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PMID:The smallest carbamoyl-phosphate synthetase. A single catalytic subdomain catalyzes all three partial reactions. 936 Oct 5

Genes for carbamoyl-phosphate synthetase II (CPS II), the first enzyme of de novo pyrimidine biosynthesis, were cloned from kinetoplastids, Trypanosoma cruzi and Leishmania mexicana. T. cruzi CPS II gene encodes a protein of 1524 amino acids that encompasses the glutaminase and CPS domains, but incorporates neither aspartate carbamoyltransferase nor dihydroorotase. The residue corresponding to lysine 993 of Escherichia coli CPS, a residue that characterizes the CPS inhibited by UMP and that is replaced by tryptophan in those inhibited by UTP, is in kinetoplastids a hydrophilic glutamine, in line with the preferential inhibition by UDP of kinetoplastid CPS II.
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PMID:Carbamoyl-phosphate synthetase II in kinetoplastids. 974 64

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

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.
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PMID:Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad). 1049 79

The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.
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PMID:An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia. 1072 15

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.
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PMID:Mechanism for acivicin inactivation of triad glutamine amidotransferases. 1117 Apr 8


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