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 activated CO2 intermediate formed in the reaction catalyzed by glutamine-dependent carbamyl phosphate synthetase was identified as carbonic-phosphoric anhydride through the use of two independent procedures. The carboxy phosphate intermediate was reduced to formate by treatment with potassium borohydride. Although both free CO2 and the enzyme-bound activated CO2 are reduced to formic acid by borohydride, it was possible to selectively introduce a 14C label into the enzyme-bound activated CO2 and thus into the formic acid derived from it. Such [14C]formate formation required the presence of ATP, KCl, and the enzyme, and evidence was obtained that the [14C]formate found is not derived from carbamyl phosphate or from bicarbonate bound nonspecifically to the enzyme. When the enzyme was treated with L-2-amino-4-oxo-5-chloropentanoate (or cyanate), the formation of [14C]formate was increased about 2-fold, a finding consistent with the previous observation that such treatment effects a similar increase in the bicarbonate-dependent cleavage of ATP catalyzed by the enzyme. When reaction mixtures containing the enzyme, [gamma-32P]ATP, and [14C]bicarbonate were methylated by treatment with diazomethane, a labeled compound was formed which cochromatographed with authentic trimethyl carboxy phosphate. Equimolar quantities of 14C and 32P wer incorporated into the intermediate, thus confirming its identification as carboxy phosphate. Nonenzymatic transphosphorylation from ATP to bicarbonate to form carboxy phosphate was also detected by diazomethane trapping.
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PMID:Identification of enzyme-bound activated CO2 as carbonic-phosphoric anhydride: isolation of the corresponding trimethyl derivative from the active site of glutamine-dependent carbamyl phosphate synthetase. 18 54

The purimidine-3 locus of Neurospora crassa specifies two enzyme activities, pyrimidine-specific carbamyl phosphate synthetase (CPSpyr) and aspartate transcarbamylase (ATC). ATC is translationally distal. CPSpyr, but not ATC, is subject to feedback inhibition by uridine triphosphate (UTP). To investigate the location of the feedback-specific region within the locus, inhibition of a number of pyr-3 alleles by UTP was investigated. All CPS+ ATC- polar alleles, revertants of CPS- ATC- polar alleles, and 5-fluorouracil-resistant mutants had normal UTP response. The location of the feedback-specific region is in or close to the CPS-specific region.
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PMID:The location of the feedback-specific region with the pyrimidine-3 locus of Neurospora crassa. 18 23

Application of the pulse-chase procedure to study of the binding and utilization of ATP by glutamine-dependent carbamyl phosphate synthetase from Escherichia coli showed that the enzyme binds the two molecules of ATP used in this reaction at the same time, and that the two ATP-binding sites are functionally different. Thus, ATP bound to the first ATP site is used for carboxy phosphate formation, and ATP bound to the second ATP site is used for phosphorylation of carbamate. The present and previous findings support a mechanism that involves intermediate formation of two highly unstable intermediates: carboxy phosphate and carbamate. It is proposed that the presence of all of the reactants on the enzyme at the start of the catalytic cycle allows immediate utilization of these labile compounds in the carbamyl phosphate synthesis reaction.
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PMID:Mechanism of the reaction catalyzed by carbamyl phosphate synthetase. Binding of ATP to the two functionally different ATP sites. 20 98

The reaction of phenylglyoxal with two enzymes in which ATP plays a complex role has been studied. Both ovine brain glutamine synthetase and Escherichia coli carbamyl phosphate synthetase [carbamoyl-phosphate synthase (glutamine); ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring); EC 2.7.2.9]were inactivated by phenylglyoxal. The specificity of this reagent for arginyl residues of the two proteins was confirmed by amino acid analysis. ATP, but not the other substrates, protected these enzymes against inactivation by phenylglyoxal. Carbamyl phosphate synthetase was also protected by IMP and ornithine, positive allosteric effectors that alter the enzymatic activity be increasing the affinity for ATP. UMP, a negative allosteric effector that decreases the affinity for ATP, did not protect against inactivation. Differential labeling experiments with [14C]phenylglyoxal showed that the number of arginyl residues protected by ATP corresponded quite well to the known number of ATP catalytic sites for each protein. These data indicate that arginyl residues at the active sites of glutamine synthetase and carbamyl phosphate synthetase are involved in the binding of ATP. This phenylglyoxal inactivation study also provided information about the mechanistic role of ATP in the two synthetases. The data obtained on glutamine synthetase support the theory that ATP is attached to the enzyme as a portion of the catalytic site, and that its presence is essential for the binding of glutamate and glutamine. The data obtained on carbamyl phosphate synthetase are consistent with the previous proposal that carbonyl phosphate is an intermediate in the ATP-dependent activation of bicarbonate by this enzyme. It is also of interest that, with both glutamine synthetase and carbamyl phosphate synthetase, only a small portion of the total arginyl population of these enzymes reacted with phenylglyoxal. A summary of previous studies on the modification of enzyme arginyl residues is presented.
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PMID:Functional arginyl residues as ATP binding sites of glutamine synthetase and carbamyl phosphate synthetase. 24 Oct 76

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.
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PMID:Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. 114 71

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

The CAD multidomain protein, which includes active sites of carbamyl phosphate synthetase II (CPS II, glutamine-dependent), aspartate transcarbamylase, and dihydroorotase, was immunostained in normal rat brains, the gliotic brains of myelin-deficient mutant rats, and brains from normal weanling hamsters. In each of these tissues CAD was observed in cells resembling astrocytes. In hamster brain, CAD immunofluorescence was also found in cells closely related to astrocytes, i.e., the Bergmann glia in cerebellum and the tanycytes surrounding the third ventricle. The astrocytic identity of the CAD-positive cells in rat brain was confirmed by double immunofluorescence staining with antibodies against glial fibrillary acidic protein (GFAP). The two enzymes carbonic anhydrase and glutamine synthetase occur in the cytoplasm of normal astrocytes in gray matter and of reactive astrocytes during gliosis. Products of each enzyme, i.e., bicarbonate and glutamine, are required for the CPS II reaction, which is the first step in the biosynthesis of pyrimidines. Therefore, the present results suggest roles for carbonic anhydrase and glutamine synthetase, as well as CAD, in pyrimidine biosynthesis in brain and a role for the astrocytes in the de novo synthesis of pyrimidines.
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PMID:Localization of the multifunctional protein CAD in astrocytes of rodent brain. 167 39

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 effects of polyamines were studied on carbamoyl-phosphate synthase II (EC 6.3.5.5.) which is the first and rate limiting enzyme in mammalian pyrimidine synthesis. Polyamines in physiological concentrations (0.1-1 mM) strongly inhibited the carbamoyl-phosphate synthesis. Of the polyamines tested spermine was the most effective followed by spermidine and putrescine. Spermine increased the KM for ATP and the requirement for Mg++ of carbamoyl-phosphate synthase reaction. UTP, an inhibitor, had similar, while phosphoribosyl-pyrophosphate, an activator of the enzyme had an opposite effect. Increasing concentrations of phosphoribosyl-pyrophosphate completely reversed the inhibition caused by spermine, while did not influence the degree of inhibition caused by UTP. A possible physiological role of polyamines in synchronizing the substrate and activator functions of phosphoribosyl-pyrophosphate in pyrimidine synthesis is suggested.
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PMID:Effect of polyamines on the carbamoyl-phosphate synthase activity of CAD protein. 248 15


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