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)

Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1). endogenous ammonia production was drastically reduced and (2). endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air. Upon re-immersion, the urea excretion rate increased 22-fold compared with that of the control specimen, which is the greatest increase among fish during emersion-immersion transitions and suggests that P. dolloi possesses transporters that facilitate the excretion of urea in water.
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PMID:Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure. 1296 53

This study aimed to elucidate the strategies adopted by the African slender lungfish, Protopterus dolloi, to ameliorate the toxicity of ammonia during short (6 days) or long (40 days) periods of aestivation in a layer of dried mucus in open air in the laboratory. Despite decreases in rates of ammonia and urea excretion, the ammonia content in the muscle, liver, brain and gut of P. dolloi remained unchanged after 6 days of aestivation compared with the control fasted for 6 days. For specimens aestivated for 40 days, the ammonia contents in the muscle, liver and gut were significantly lower than those of the control fasted for 40 days, which suggests a decrease in the rate of ammonia production. In addition, there were significant increases in contents of alanine, aspartate and glutamate in the muscle, which suggests decreases in their catabolism. During the first 6 days and the last 34 days of aestivation, the rate of ammonia production was reduced to 26% and 28%, respectively, of the control rate (6.83 micromol day(-1) g(-1) on day 0). During the first 6 days and the next 34 days of aestivation, the averaged urea synthesis rate was 2.39-fold and 3.8-fold, respectively, greater than the value of 0.25 micromol day(-1) g(-1) for the day 0 control kept in water. No induction of activities of the ornithine-urea cycle (OUC) enzymes was observed in specimens aestivated for 6 days, because the suppression of ammonia production led to a light demand on the OUC capacity. For specimens aestivated for 40 days, the activities of carbamoyl phosphate synthetase, ornithine transcarbamylase and argininosuccinate synthetase + lyase were significantly greater than those of the control fasted for 40 days. This is in agreement with the observation that the rate of urea synthesis in the last 34 days was greater than that in the first 6 days of aestivation. P. dolloi aestivated in a thin layer of dried mucus in open air with high O(2) tension throughout the 40 days of aestivation, which could be the reason why it was able to sustain a high rate of urea synthesis despite this being an energy-intensive process. Our results indicate that a reduction in ammonia production and decreases in hepatic arginine and cranial tryptophan contents are important facets of aestivation in P. dolloi.
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PMID:Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land. 1474 10

The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli revealed the existence of a molecular tunnel that has been proposed to facilitate the translocation of reaction intermediates between remotely located active sites. Five highly conserved glutamate residues, including Glu-25, Glu-383, Glu-577, Glu-604, and Glu-916, are close together in two clusters in the interior wall of the molecular tunnel that enables the intermediate carbamate to migrate from the site of synthesis to the site of utilization. Two arginines, Arg-306 and Arg-848, are located at either end of the carbamate tunnel and participate in the binding of ATP at each of the two active sites within the large subunit of CPS. The mutation of Glu-25 or Glu-577 results in a diminution in the overall rate of carbamoyl phosphate formation. Similar effects are observed upon mutation of Arg-306 and Arg-848 to alanine residues. The conserved glutamate and arginine residues may function in concert with one another to control entry of carbamate into the tunnel prior to phosphorylation to carbamoyl phosphate. The electrostatic environment of tunnel interior may help to stabilize the tunnel architecture and prevent decomposition of carbamate through protonation.
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PMID:Access to the carbamate tunnel of carbamoyl phosphate synthetase. 1508 91

Carbamoyl phosphate synthetase plays a key role in both pyrimidine and arginine biosynthesis by catalyzing the production of carbamoyl phosphate from one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine. The enzyme from Escherichia coli consists of two polypeptide chains referred to as the small and large subunits, which contain a total of three separate active sites that are connected by an intramolecular tunnel. The small subunit harbors one of these active sites and is responsible for the hydrolysis of glutamine to glutamate and ammonia. The large subunit binds the two required molecules of MgATP and is involved in assembling the final product. Compounds such as L-ornithine, UMP, and IMP allosterically regulate the enzyme. Here, we report the three-dimensional structure of a site-directed mutant protein of carbamoyl phosphate synthetase from E. coli, where Cys 248 in the small subunit was changed to an aspartate. This residue was targeted for a structural investigation because previous studies demonstrated that the partial glutaminase activity of the C248D mutant protein was increased 40-fold relative to the wild-type enzyme, whereas the formation of carbamoyl phosphate using glutamine as a nitrogen source was completely abolished. Remarkably, although Cys 248 in the small subunit is located at approximately 100 A from the allosteric binding pocket in the large subunit, the electron density map clearly revealed the presence of UMP, although this ligand was never included in the purification or crystallization schemes. The manner in which UMP binds to carbamoyl phosphate synthetase is described.
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PMID:Long-range allosteric transitions in carbamoyl phosphate synthetase. 1532 82

Carbamoyl phosphate synthetase I (CPSI) deficiency, a recessively inherited error of the urea cycle, causes life-threatening hyperammonaemia. CPSI is a multidomain 1500-residue liver mitochondrial matrix protein that is allosterically activated by N-acetyl-l-glutamate, and which synthesises carbamoyl phosphate (CP) in three steps: bicarbonate phosphorylation by ATP, carbamate synthesis from carboxyphosphate and ammonia, and carbamate phosphorylation by ATP. Several missense mutations of CPSI have been reported in patients with CPSI deficiency, but the actual pathogenic potential and effects on the enzyme of these mutations remain non-characterised. Since the structure of Escherichia coli CPS is known and systems for its overexpression and purification are available, we have constructed and purified eight site-directed mutants of E.coli CPS affecting the enzyme large subunit (A126M, R169H, Q262P, N301K, P360L, V640R, R675L, S789P) that are homologous to corresponding missense mutations found in patients with CPSI deficiency, studying their stability and their ability to catalyse the CPS reaction as well as the partial reactions that reflect the different reactional steps, and analysing the substrate kinetics for the overall and partial reactions. The results show that all the mutations significantly decrease CP synthesis without completely inactivating the enzyme (as reflected in the catalysis of at least one partial reaction), that one of these mutations (Q262P) causes marked enzyme instability, and validate the use of E.coli CPS as a pathogenicity testing model for CPSI deficiency. The causality of the reported clinical mutations is supported and the derangements caused by the mutations are identified, revealing the specific roles of the residues that are mutated. In particular, the findings highlight the importance for carbamate phosphorylation and for allosteric activation of a loop that coordinates K(+), stress the key role of intersubunit interactions for CPS stability, and suggest that lid opening at both phosphorylation sites is concerted.
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PMID:Understanding carbamoyl phosphate synthetase deficiency: impact of clinical mutations on enzyme functionality. 1587 73

Using the genomic SELEX, a total of six Escherichia coli DNA fragments have been identified, which formed complexes with transcription factor RutR. The RutR regulon was found to include a large number of genes encoding components for not only degradation of pyrimidines but also transport of glutamate, synthesis of glutamine, synthesis of pyrimidine nucleotides and arginine, and degradation of purines. DNase I footprinting indicated that RutR recognizes a palindromic sequence of TTGACCAnnTGGTCAA. The RutR box in P1 promoter of carAB encoding carbamoyl phosphate synthetase, a key enzyme of pyrimidine synthesis, overlaps with the PepA (CarP) repressor binding site, implying competition between RutR and PepA. Adding either uracil or thymine abolished RutR binding in vitro to the carAB P1 promoter. Accordingly, in the rutR-deletion mutant or in the presence of uracil, the activation in vivo of carAB P1 promoter was markedly reduced. Northern blot analysis of the RutR target genes indicated that RutR represses the Gad system genes involved in glutamate-dependent acid resistance and allantoin degradation. Altogether we propose that RutR is the pyrimidine sensor and the master regulator for a large set of the genes involved in the synthesis and degradation of pyrimidines.
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PMID:RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimidines. 1791 80

The first part of this review is concerned with the balance between N input and output as urinary urea. I start with some observations on classical biochemical studies of the operation of the urea cycle. According to Krebs, the cycle is instantaneous and automatic, as a result of the irreversibility of the first enzyme, carbamoyl-phosphate synthetase 1 (EC 6.3.5.5; CPS-I), and it should be able to handle many times the normal input to the cycle. It is now generally agreed that acetyl glutamate is a necessary co-factor for CPS-1, but not a regulator. There is abundant evidence that changes in dietary protein supply induce coordinated changes in the amounts of all five urea-cycle enzymes. How this coordination is achieved, and why it should be necessary in view of the properties of the cycle mentioned above, is unknown. At the physiological level it is not clear how a change in protein intake is translated into a change of urea cycle activity. It is very unlikely that the signal is an alteration in the plasma concentration either of total amino-N or of any single amino acid. The immediate substrates of the urea cycle are NH3 and aspartate, but there have been no measurements of their concentration in the liver in relation to urea production. Measurements of urea kinetics have shown that in many cases urea production exceeds N intake, and it is only through transfer of some of the urea produced to the colon, where it is hydrolysed to NH3, that it is possible to achieve N balance. It is beginning to look as if this process is regulated, possibly through the operation of recently discovered urea transporters in the kidney and colon. The second part of the review deals with the synthesis and breakdown of protein. The evidence on whole-body protein turnover under a variety of conditions strongly suggests that the components of turnover, including amino acid oxidation, are influenced and perhaps regulated by amino acid supply or amino acid concentration, with insulin playing an important but secondary role. Molecular biology has provided a great deal of information about the complex processes of protein synthesis and breakdown, but so far has nothing to say about how they are coordinated so that in the steady state they are equal. A simple hypothesis is proposed to fill this gap, based on the self-evident fact that for two processes to be coordinated they must have some factor in common. This common factor is the amino acid pool, which provides the substrates for synthesis and represents the products of breakdown. The review concludes that although the achievement and maintenance of N balance is a fact of life that we tend to take for granted, there are many features of it that are not understood, principally the control of urea production and excretion to match the intake, and the coordination of protein synthesis and breakdown to maintain a relatively constant lean body mass.
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PMID:The mysteries of nitrogen balance. 1908 45

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.
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PMID:Assimilation of excess ammonium into amino acids and nitrogen translocation in Arabidopsis thaliana--roles of glutamate synthases and carbamoylphosphate synthetase in leaves. 1955 10

NAG (N-acetyl-L-glutamate), the essential allosteric activator of the first urea cycle enzyme, CPSI (carbamoyl phosphate synthetase I), is a key regulator of this crucial cycle for ammonia detoxification in animals (including humans). Automated cavity searching and flexible docking have allowed identification of the NAG site in the crystal structure of human CPSI C-terminal domain. The site, a pocket lined by invariant residues and located between the central beta-sheet and two alpha-helices, opens at the beta-sheet C-edge and is roofed by a three-residue lid. It can tightly accommodate one extended NAG molecule having the delta-COO- at the pocket entry, the alpha-COO- and acetamido groups tightly hydrogen bonded to the pocket, and the terminal methyl of the acetamido substituent surrounded by hydrophobic residues. This binding mode is supported by the observation of reduced NAG affinity upon mutation of NAG-interacting residues of CPSI (recombinantly expressed using baculovirus/insect cells); by the fine-mapping of the N-chloroacetyl-L-glutamate photoaffinity labelling site of CPSI; and by previously established structure-activity relationships for NAG analogues. The location of the NAG site is identical to that of the weak bacterial CPS activator IMP (inosine monophosphate) in Escherichia coli CPS, indicating a common origin for these sites and excluding any relatedness to the binding site of the other bacterial CPS activator, ornithine. Our findings open the way to the identification of CPSI deficiency patients carrying NAG site mutations, and to the possibility of tailoring the activator to fit a given NAG site mutation, as exemplified here with N-acetyl-L(+/-)-beta-phenylglutamate for the W1410K CPSI mutation.
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PMID:Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase. 1975 28

This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.
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PMID:The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water. 1991 25


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