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

---

Query: EC:6.3.5.5 (CPS)
1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
...
PMID:Kinetic mechanism of Escherichia coli carbamoyl-phosphate synthetase. 21 4

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.
...
PMID:Functional arginyl residues as ATP binding sites of glutamine synthetase and carbamyl phosphate synthetase. 24 Oct 76

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.
...
PMID:Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis. 44 63

Two patients presenting with acute fatty liver of pregnancy were studied. Because of similarities between acute fatty liver of pregnancy and Reye's syndrome, we investigated hepatic ultrastructure, urea-cycle enzyme activities, and plasma amino acids. Initial liver biopsies obtained 12 and 21 days after the onset of illness demonstrated microvesicular fat deposition and mitochondrial ultrastructural changes, including pleomorphism and abundant crystalline inclusions. In both biopsies, activity of the mitochondrial urea-cycle enzyme OTC was markedly below normal limits. Activity of the other mitochondrial urea-cycle enzyme, CPS, was low in one patient. Abnormalities of these enzymes persisted in second biopsies obtained at 9 and 28 weeks, respectively. By 44 weeks all urea-cycle enzyme activities had returned to normal in one patient. However, in the other patient OTC activity was still reduced at 52 weeks, although it had doubled in comparison to previous biopsies. Morphological changes of the mitochondria generally improved in parallel with the urea-cycle enzymes. Plasma amino acids, obtained at the time of the initial biopsies, demonstrated a generalized hypoaminoacidemia with the exception of glutamate. Serial observations in patients with this rare disease indicate that there are similarities with Reye's syndrome, in particular, reduced activity of the mitochondrial urea-cycle enzymes. But there are important differences. (1) Enzymatic and ultrastructural abnormalities of mitochondria persist for a longer period of time than in Reye's syndrome. (2) Mitochondrial ultrastructure is different. (3) Plasma amino acid profiles are different.
...
PMID:Abnormalities of hepatic mitochondrial urea-cycle enzyme activities and hepatic ultrastructure in acute fatty liver of pregnancy. 46 76

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of N-acetyl-L-glutamate, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well in parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in N-acetyl-L-glutamate concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.
...
PMID:Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes. 50 1

Synthesis of glucose from lactate and generation of urea from ammonia were inhibited when sodium benzoate was added to suspensions of rat hepatocytes. Assays with isolated mitochondria suggested pyruvate carboxylase and the N-acetyl-L-glutamate (NAG)-dependent carbamoylphosphate synthetase (CPS-I) as potential sites of inhibition for both pathways, owing to a shared dependency on aspartate efflux from the mitochondria and its subsequent conversion to oxaloacetate in the cytosol. Assays with isolated hepatocytes indicated inhibition to be initiated by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. Measurements of adenine nucleotides showed that benzoate metabolism did not sufficiently alter energy status to account for the observed inhibition. Consistent with these interpretations, acceleration of the conversion of benzoyl CoA to hippurate by the addition of glycine restored the levels of free CoA and acetyl CoA and the rates of gluconeogenesis and ureagenesis. Reduction of the levels of aspartate and glutamate, presumably by interference with the anapleurotic function of pyruvate carboxylase, most likely accounted for inhibition of gluconeogenesis by benzoate. Whether reduced flux through the urea cycle also contributed to inhibition of gluconeogenesis (by diminishing cytosolic conversion of aspartate to oxaloacetate) requires further study. Depression of glutamate and acetyl CoA to levels at or below the Km for NAG synthetase probably accounted for the observed inhibition of ureagenesis. Rates of urea production were observed to vary with changes in the levels of NAG, suggesting NAG-dependent CPS-I to be the primary site of inhibition of ureagenesis by benzoate.
...
PMID:On the mechanism of inhibition of gluconeogenesis and ureagenesis by sodium benzoate. 167 73

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200-300 times lower than the levels of N-acetyl-L-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 micrograms/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-L-aspartyl-L-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.
...
PMID:N-acetyl-L-glutamate in brain: assay, levels, and regional and subcellular distribution. 194 68

N-Acetyl-L-glutamate has been examined with regard to its ability to activate carbamoyl phosphate synthetase I (EC 6.3.4.16). Substance(s) inhibitory to carbamoyl phosphate synthetase, present even in the partially purified preparation of rat liver extracts, interfered with the measurement of acetylglutamate. In the experiments using chelating agents, metals were apparently involved in this inhibition. When the partially purified preparation of liver extract was placed on a Chelex 100 column, the inhibitor was eliminated and accurate measurements of acetylglutamate content could be made. Evidence supporting the validity of this improved method is given. A significant difference was observed between acetylglutamate levels determined by the present method and by the one using aminoacylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) to hydrolyze acetylglutamate followed by assay of the glutamate generated. We searched for the presence of glutamate derivatives other than acetylglutamate. When impure tissue preparations containing acetylglutamate were treated with a commercial preparation of aminoacylase, there was an excess amount of glutamate apparently derived from compounds other than acetylglutamate. This can lead to an overestimation of the tissue levels of acetylglutamate.
...
PMID:An improved method for determination of N-acetyl-L-glutamate by its function as an activator of carbamoyl phosphate synthetase I. 230 60

The early enzymes of arginine biosynthesis in Neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline. The final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm. We have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract. Acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) were purified to homogeneity and near homogeneity, respectively. The large catalytic subunit of carbamoyl-phosphate synthetase was also purified to homogeneity. The three enzymes were resolved into separate fractions by chromatography on three dye-ligand affinity resins, which are specific for nucleotide binding enzymes and have a high protein binding capacity. High performance liquid chromatography was employed in the final stages of purification and was extremely effective in fractionating both acetylglutamate kinase and acetylglutamyl-phosphate reductase from proteins with very similar properties, which were not removed by other techniques. The purified proteins were used to raise specific antisera against these proteins. Acetylglutamate kinase and acetylglutamyl-phosphate reductase were shown to be immunologically unrelated. This finding suggests that the arg-6 locus encompasses two nonoverlapping cistrons. The antisera raised against carbamoyl-phosphate synthetase has been shown to cross-react with related enzymes in Saccharomyces cerevisiae, Escherichia coli, and rat liver (Ness, S. A., and Weiss, R. L. (1985) J. Biol. Chem. 260, 14355-14362). Acetylglutamate kinase is a regulatory enzyme and has been shown to be feedback-inhibited by arginine. We have determined the submitochondrial localization of acetylglutamate kinase and the second arg-6 product, acetylglutamyl-phosphate reductase. Both enzymes were shown to be soluble matrix enzymes. We discuss the relevance of this finding with respect to possible mechanisms for end product inhibition of a mitochondrial enzyme by a cytoplasmic effector.
...
PMID:Simultaneous purification of three mitochondrial enzymes. Acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from Neurospora crassa. 242 Jul 93

Increased blood ammonia was induced in fasting mice by ip administration of 200 mg/kg Na-valproate followed 1 h later by 13 and 4 mmol/kg alanine and ornithine, respectively. When valproate was not used blood or liver ammonia was not increased, but increases were observed in liver glutamate (5-fold), glutamine (2-fold), aspartate (5-fold), acetylglutamate (15-fold), citrulline (35-fold), argininosuccinate (11-fold), arginine (11-fold), and urea (3-fold). The level of carbamoyl phosphate (less than 2 nmol/g) was, by far, the lowest of all urea cycle intermediates. The large increase in citrulline indicates that argininosuccinate synthesis was limiting, and that the increase in acetylglutamate induced a considerable activation of carbamoyl phosphate synthetase, which agrees with theoretical expectations, irrespective of the actual KD value for acetylglutamate. Pretreatment with valproate resulted in lower hepatic levels of glutamate, glutamine, aspartate, acetyl-CoA, and acetylglutamate. At the level found of acetylglutamate the activation of carbamoyl phosphate synthetase would be expected to be similar to that without valproate. Indeed, the levels of citrulline were similar with or without valproate. Argininosuccinate, arginine, and urea levels exhibited little if any change. Although the model used may not replicate exactly the situation in patients, from our results it appears that changes in citrullinogenesis or in other steps of the urea cycle do not account for the increase in blood ammonia induced by valproate, and it is proposed that valproate may alter glutamine metabolism.
...
PMID:Changes in urea cycle-related metabolites in the mouse after combined administration of valproic acid and an amino acid load. 250 68


1 2 3 4 5 6 7 Next >>

---