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 arginine-specific carbamoyl-phosphate synthase of yeast was stabilized sufficiently to allow partial purification of the enzyme (30- to 40-fold). The synthase (mol. wt 115000) comprised two unequal subunits: a heavy subunit (mol. wt 80000) capable of catalysing synthesis of carbamoyl phosphate with ammonia as a nitrogen donor and a light subunit conferring upon the holoenzyme the ability to utilize glutamine. The enzyme had unusually high affinity for ATP (Km = 0.2 mM) and atypical negative cooperativity for glutamine binding ([S]0.5 = 0.25 mM). Glutamine activity was not modulated by possible effectors such as arginine, ornithine or N-acetylglutamate. Thus, although the yeast arginine enzyme physically and functionally resembles the single enteric synthase, the systems differ substantially both in kinetic properties and in regulation of activity.
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PMID:Purification and properties of the arginine-specific carbamoyl-phosphate synthase from Saccharomyces cerevisiae. 20 52

The kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase has been determined at pH 7.5, 25 degrees C. With ammonia as the nitrogen source, the initial velocity and product inhibition patterns are consistent with the ordered addition of MgATP, HCO3-, and NH3. Phosphate is then released and the second MgATP adds to the enzyme, which is followed by the ordered release of MgADP, carbamoyl phosphate, and MgADP. With glutamine as the ammonia donor, the patterns are consistent with a two-site mechanism in which glutamine binds randomly to the small molecular weight subunit producing glutamate and ammonia. Glutamate is released and the ammonia is transferred to the larger subunit. Carbamoyl-phosphate synthetase has also been shown to require a free divalent cation for full activity.
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PMID:Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase. 21 4

1. Ammonia liberated continuously in large amounts in muscle, kidney and brain is used immediately for the synthesis of mainly glutamine because of the toxic effects of elevated ammonia concentrations. After glutamine hydrolysis in the liver ammonia serves as substrate for the urea biosynthesis. In ureotelic animals urea is the quantitatively most important product for the elimination of surplus nitrogen. 2. The rate of urea biosynthesis depends on the amount of surplus nitrogen and acts as regulatory factor for the nitrogen balance of the adult organism. 3. Urea cycle abnormalities in liver diseases or inborn enzymatic defects are important factors leading to hyperammonaemia in patients. 4. The hyperammonaemia induces an increase of the rate of hepatic pyrimidine nucleotide biosynthesis as a consequence of an ineffective feedback inhibition of the glutamine-dependent carbamoyl phosphate synthetase. 5. The distribution of ammonia between intra- and extracellular space and the amount of ammonium ions excreted in the urine depend on the pH value. An alkalosis induces an intracellular ammonia load and inhibits the urinary ammonium ion excretion, which is increased in acidosis as one mechanism of protein elimination. 6. The ammonia-induced inhibition of the citric acid cycle by an alpha-ketoglutarate deficiency is one important reason for the neurotoxicity of ammonia, which is the main point in the pathogenesis of hepatic coma.
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PMID:[Biochemical and pathophysiological aspects of hyperammonaemia (author's transl)]. 31 94

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

The effects of unilateral nephrectomy (UN) and streptozotocin (STZ) diabetes on the activities of enzymes involved in uridine and cytidine synthesis in early renal growth (3-14 days after stimulus to growth) have been compared. Measurements were also made of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) and of glucose 6-phosphate (G6P), UDP-glucose, and glycogen, in relation to phosphoribosyl pyrophosphate, ribonucleotide, and complex carbohydrate formation. There were striking differences in the activities of CTP synthetase, G6PDH, and 6PGDH in the two conditions, with a three-fold increase in all three enzymes at 3 and 5 days and a two-fold increase above basal values at 14 days of STZ diabetes. The UN group showed no significant change in CTP synthetase at any stage and the activity of G6PDH and 6PGDH only kept pace with renal growth. Changes in routes of uridine synthesis were less marked, with a more rapid rise in carbamoyl-phosphate synthetase (glutamine) and a lesser response of dihydroorotate dehydrogenase in the UN relative to the STZ-diabetic groups. The enzymes of complex II and of uracil phosphoribosyltransferase showed essentially similar patterns during renal hypertrophy in UN and STZ diabetes. The parallel increase in CTP synthetase, G6PDH, and 6PGDH in the kidney in diabetes, also known to increase in growth situations in hepatomas and in renal tumors, is discussed in relation to hormone signals involved in renal growth. The importance of the concentration of CTP, and thus of CTP synthetase, in the CTP-cytidyltransferase reaction, an enzyme with a high Km for CTP, makes the present observation of the striking increase in CTP synthetase in STZ diabetes of particular interest in relation to phosphatidylcholine formation and hormone signal transduction.
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PMID:Uridine and cytidine nucleotide synthesis in renal hypertrophy: biochemical differences in response to the growth stimulus of diabetes and unilateral nephrectomy. 138 Dec

Measurements have been made of the activities of the enzymes of the de novo and salvage pathways of pyrimidine synthesis (carbamoyl phosphate synthetase II (glutamine) (EC 6.3.5.5); dihydroorotate dehydrogenase (EC 1.3.99.11); the overall activity of Complex II (orotate phosphoribosyl pyrophosphate transferase (EC 2.4.2.10) and orotidine 5-phosphate decarboxylase (EC 4.1.1.23); uracil phosphoribosyltransferase (EC 2.4.2.9)) in the mammary gland of rats at different stages of the lactation cycle and the effects of diabetes on the activity of these enzymes in lactation have been studied. From a consideration of the changes in enzyme activities and the changes in the tissue concentration of phosphoribosyl pyrophosphate, an activator of the de novo pathway and substrate for both the de novo and salvage routes, it is concluded that the de novo pathway is the major route of pyrimidine synthesis in mammary tissue. Diabetes decreases the activity of the enzymes of the de novo pathway; the effects are particularly marked for Complex II. The present results on pyrimidine synthesis are compared to the pattern for purine synthesis previously published.
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PMID:Pyrimidine nucleotide synthesis in the rat mammary gland: changes in the lactation cycle and effects of diabetes. 147 92

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

Biotin carboxylase [biotin-carboxyl-carrier-protein:carbon-dioxide ligase (ADP-forming), EC 6.3.4.14] is the enzyme mediating the first step of the acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] reaction. We screened an Escherichia coli DNA library and a DNA fragment carrying the biotin carboxylase gene fabG, and its flanking regions were cloned. The gene for biotin carboxyl carrier protein was found 13 base pairs upstream of the fabG gene. Nucleotide sequencing of the recombinant plasmids revealed that the fabG codes for a 449-amino acid residue protein with a calculated molecular weight of 49,320, a value in good agreement with that of 51,000 determined by SDS/polyacrylamide gel electrophoresis of the purified enzyme. The deduced amino acid sequence of biotin carboxylase is also consistent with the partial amino acid sequence determined by Edman degradation. The primary structure of this enzyme exhibits a high homology with those of other biotin-dependent enzymes and carbamoyl-phosphate synthetase [carbon-dioxide:L-glutamine amino-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5]; therefore, all these enzymes probably function through the same mechanism of reaction.
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PMID:Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit. 168 20

The change in reaction energetics of the bicarbonate-dependent ATPase reaction of Escherichia coli carbamoyl phosphate synthetase has been investigated for two site-directed mutations of the essential cysteine in the small subunit. Cysteine 269 has been proposed to facilitate the hydrolysis of glutamine by the formation of a glutamyl-thioester intermediate. The two mutant enzymes, C269S and C269G, along with the isolated large subunit, exhibit a 2-2.6-fold increase in the bicarbonate-dependent ATPase reaction relative to that observed for the wild type enzyme. In the presence of glutamine the overall enhancement is 3.7 and 9.0 for the C269G and C269S mutant enzymes, respectively. Carboxyphosphate is an intermediate in the bicarbonate-dependent ATPase reaction. The cause of the rate enhancements was investigated by measuring the positional isotope exchange rate in [gamma-18O4] ATP relative to the net rate of ATP hydrolysis. This ratio (Vex/Vchem) is a measure of the partitioning of the enzyme-carboxyphosphate-ADP complex. The partitioning ratio for the mutants is identical within experimental error to that observed for the wild type enzyme. This observation is consistent with the conclusion that the ground state for the enzyme-carboxyphosphate-ADP complex in the mutants is destabilized relative to the same complex in the wild type enzyme. If the increase in the absolute rate of ATP hydrolysis was due to a stabilization of the transition state for carboxyphosphate hydrolysis then the positional isotope exchange rate relative to the chemical hydrolysis rate would have been expected to decrease in the mutants.
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PMID:Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group. 182 18


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