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)

Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide.
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PMID:Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain. 274 25

The carAB operon of Salmonella typhimurium encoding carbamoyl-phosphate synthetase (CPSase) has been cloned, and the nucleotide sequence of the first gene of the operon, carA, together with 760 base pairs of the 5'-flanking region was determined. The product of the carA gene is the small subunit of CPSase. It catalyzes the transfer of the amide group from glutamine to an NH3-site on the heavy subunit. Primer extension and S1 nuclease mapping of in vivo carAB transcripts revealed that transcription is similar to that of Escherichia coli [Piette, J. et al. (1984) Proc. Natl Acad. Sci. USA 81, 4134-4138] in its initiation at two promoters, P1 and P2, controlled by pyrimidines and arginine, respectively. The arginine control is mediated through binding to the arginine repressor (argR). The involvement of titratable regulatory elements is indicated by the escape from both arginine and pyrimidine control, when the operon is present in multicopies on a plasmid. Measurements of CPSase levels in mutants which allows independent manipulation of the intracellular uracil and cytosine nucleotide pools show, that both uracil and cytosine nucleotides are required for full repression and that limitation of either nucleotide results in derepression of CPSase synthesis. Deletion analyses indicate that regions upstream of the P1 promoter are required for normal expression from this promoter but not from P2.
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PMID:Nucleotide sequence of the carA gene and regulation of the carAB operon in Salmonella typhimurium. 284 75

Ammonia assimilation for urea synthesis by liver mitochondria in marine elasmobranchs involves, initially, formation of glutamine which is subsequently utilized for mitochondrial carbamoyl phosphate synthesis [P. M. Anderson and C. A. Casey (1984) J. Biol. Chem. 259, 456-462]. The purpose of this study was to determine if the glutamine synthetase catalyzing this first step in urea synthesis has properties uniquely related to this function. Glutamine synthetase has been highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme has a molecular weight of approximately 400,000 in the presence of Mg2+, MgATP, and L-glutamate, but dissociates reversibly to a species with a molecular weight of approximately 200,000 in the absence of MgATP and L-glutamate. Association with the glutamine- and acetylglutamate-dependent carbamoyl phosphate synthetase, also located in the mitochondria, could not be demonstrated. The subunit molecular weight is approximately 46,000. The pH optimum of the biosynthesis reaction is 7.1-7.4. The purified enzyme is stabilized by MgATP and glutamate and by ethylene glycol, and is activated by 5-10% ethylene glycol. The apparent Km values for MgATP, L-glutamate, and ammonia (NH4+-NH3) are 0.7, 11.0, and 0.015 mM, respectively. Mg2+ in excess of that required to complex ATP as MgATP is required for maximal activity; Mn2+ cannot replace Mg2+. The enzyme is activated by low concentrations of chloride, bromide, or iodide; this effect appears to be related to decreases in the apparent Km for glutamate. The enzyme is inhibited by physiological concentrations of urea, but is not significantly affected by physiological concentrations of trimethylamine-N-oxide. Except for activation by halogen anions and the very low apparent Km for ammonia, this elasmobranch glutamine synthetase has properties similar to those reported for mammalian and avian glutamine synthetases. The very low apparent Km for ammonia may be specifically related to the unique role of this glutamine synthetase in mitochondrial assimilation of ammonia for urea synthesis.
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PMID:Purification and properties of glutamine synthetase from liver of Squalus acanthias. 286 Aug 71

Acetyl-coenzyme A carboxylase (Ac-CoA carboxylase; EC 6.4.1.2) catalyzes the rate-limiting reaction in long-chain fatty acid biosynthesis. To investigate the mechanism of genetic control of expression of Ac-CoA carboxylase and the relationship between its structure and function, cDNA clones for Ac-CoA carboxylase were isolated. The complete coding sequence contains 7035 bases; it encodes a polypeptide chain of 2345 amino acids having a Mr of 265,220. The sequences of several CNBr peptides of Ac-CoA carboxylase were localized within the predicted protein sequence as were those peptides that contain the sites for phosphorylation. The deduced protein contains one putative site for biotinylation in the NH2-terminal half. The "conserved" biotinylation site peptide, Met-Lys-Met, is preceded by valine, whereas alanine is found in a similar position in all other known biotin-containing proteins. The primary sequences of Ac-CoA carboxylase and carbamoyl phosphate synthetase exhibit substantial identity.
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PMID:Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase. 290 Oct 88

Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5' proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CP A1 CP A2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH2-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO2H.
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PMID:Nucleotide sequence of the pyrimidine specific carbamoyl phosphate synthetase, a part of the yeast multifunctional protein encoded by the URA2 gene. 303 94

Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl2, NH4Cl, and the activator N-acetyl-L-glutamate. These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species and a 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH4Cl and N-acetyl-L-glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.
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PMID:Controlled proteolysis of ammonia-dependent carbamoyl-phosphate synthetase I from Syrian hamster liver. 325 64

Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.
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PMID:Domain structure of rat liver carbamoyl phosphate synthetase I. 349 Oct 68

The urea biosynthetic pathway functions in mammalian liver to convert excess ammonia to urea and to maintain the concentration of ammonia in blood at nontoxic levels. This action is accomplished by enzymatic adaptation to quantitative changes in dietary protein. The first two enzymes of the pathway are found in the intestine of the adult mouse, but they do not adapt to dietary change. The enzymes in the intestine produce citrulline, which is carried by the bloodstream to the kidney, where it is converted by the next two enzymes of the pathway to arginine. This mechanism serves as the major source of circulating arginine. We have demonstrated that, at birth, the arginine-synthesizing enzymes in the kidney of the C57Bl/6 mouse are minimally developed, whereas in the intestine activity of carbamoyl-phosphate synthase is elevated and argininosuccinate synthase and lyase, usually present only in trace quantities in the adult intestine, are markedly increased in the newborn. The arginine formed cannot be converted to urea, since arginase does not appear in intestinal cells of the mouse until the age of 15 days. Except for liver, intestine has the most rapid protein turnover of any normal tissue. Our study indicates that, at a time when no other endogenous source of arginine for protein synthesis is available, the intestine of the newborn C57Bl mouse is capable of synthesizing arginine from either citrulline or NH3 and CO2.
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PMID:Development of arginine-synthesizing enzymes in mouse intestine. 372 68

The purpose of this study was to clarify how changes in acid-base balance influence the rate of urea synthesis in vivo. Since ureagenesis was increased by an ammonium infusion into rats, regulation seemed to be a function of the blood ammonium concentration. The rate of urea synthesis was constant at a fixed rate of ammonium infusion and independent of the conjugate base infused, chloride or bicarbonate. The steady-state blood ammonium concentration was higher in the rats that developed metabolic acidosis. Thus it appeared that regulation was not directly mediated by this ammonium concentration per se. The rate of urea synthesis was also independent of the blood pH. Accordingly, the rate of urea synthesis was examined as a function of the plasma NH3 concentration. The rate of ureagenesis was found to be directly proportional to the plasma NH3 concentration. Assuming that plasma NH3 levels reflect those in mitochondria, the NH3 concentration yielding half-maximal rates of urea synthesis (close to 2 microM) was in the same range as Km for the rate-limiting step in ureagenesis, carbamoyl phosphate synthetase (EC 6.3.4.16). These results suggest that, at a constant ammonium concentration, the decreased rate of ureagenesis caused by a pH fall in vitro could reflect an acidosis-induced decline in the concentration of true substrate (NH3) for this pathway.
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PMID:Regulation of urea synthesis by acid-base balance in vivo: role of NH3 concentration. 381 36

Experiments with carbamoyl phosphate synthetase (ammonia) in solution and in isolated mitochondria are reported which show the following. NH3 rather than NH4+ is the substrate of the enzyme. The apparent Km of NH3 for the purified enzyme is about 38 microM. The apparent Km for NH3 measured in intact isolated mitochondria is about 13 microM. This value was obtained for both coupled and uncoupled mitochondria and was unchanged when the rate of carbamoyl phosphate synthesis was increased 2-fold by incubating uncoupled mitochondria in the presence of 5 mM-N-acetylglutamate. According to the literature, the concentration of NH3 in liver is well below the measured apparent Km. On the basis of this and previous work we conclude that, quantitatively, changes in liver [NH3] and [ornithine] are likely to be the most important factors in the fast regulation of synthesis of carbamoyl phosphate and urea. This conclusion is consistent with all available evidence obtained with isolated mitochondria, isolated hepatocytes, perfused liver and whole animals.
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PMID:The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ. 403 55


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