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 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

The mechanism of the reaction catalyzed by rat liver mitochondrial carbamoyl-phosphate synthetase has been studied by using [beta-18O2]ATP and HC18O-3, monitoring the isotopic composition of adenosine triphosphate (ATP) and inorganic phosphate (Pi) by high-resolution 31P NMR spectroscopy. In the presence of both HCO3- and acetylglutamate, the enzyme catalyzes the exchange of oxygen atoms between the beta, gamma bridging and the beta nonbridging positions of ATP. Addition of NH3 stops the exchange, Pi released by the ATPase activity of the enzyme in the absence of NH3 contains one oxygen atom from HC18O3- but there is no incorporation of 18O into ATP. There is no significant incorporation of [14C]ADP or 32Pi into ATP. It is concluded that in the enzyme-ATPA.HCO30.ATPB complex formed in the presence of ATP and HCO3- there is reversible transfer of the gamma-PO3 group of ATPA (the molecule that yields Pi) to HCO3- without dissociation of products. The beta-PO3 of the enzyme-bound ADP that is formed can rotate. Virtually all of the complex appears to be in the form in which ATPA is cleaved, but in the absence of NH3, ATP is reconstituted and dissociates from the complex on at least 75% of the occasions. On the remainder, the carbonyl phosphate is cleaved in an irreversible process that yields Pi and a low-energy form of carbonic acid (probably HCO3-). NH3 reacts rapidly and irreversibly with the complex, and at saturation the rate (greater than 10 times the rate of Pi release in the absence of NH3) is sufficient to prevent dissociation of ATPA. In the absence of HCO3- an enzyme-ATPA.ATPB complex is formed, but cleavage of the bond between beta, gamma bridging oxygen and P gamma of ATPA does not occur.
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PMID:Mechanism of activation of bicarbonate ion by mitochondrial carbamoyl-phosphate synthetase: formation of enzyme-bound adenosine diphosphate from the adenosine triphosphate that yields inorganic phosphate. 626 8

Two paramagnetic probes, viz., Mn2+ and Cr3+-ATP, were used to map distances to various loci on carbamoyl-phosphate synthetase by using NMR measurements. The paramagnetic influence of Mn2+ on the 1H of L-glutamate and L-ornithine was measured at 200 and 360 MHz. On the basis of these data, a correlation time for the paramagnetic interaction was determined (2 X 10(-9) s) and used to compute distances. These were in the range 7-9 A. Distances were also calculated from Mn2+ to the 13C-5 atom of glutamate (8.6 A), to the monovalent cation site (approximately 8 A), and to the phosphorus atoms of ATP in the Co(NH3)4ATP complex. For studies of the monovalent cation site relaxation rates of 6Li+, 7Li+, and 15NH4+ were measured. With Cr3+ ATP as a paramagnetic substrate analogue, Cr3+ to 13C distances were measured with the substrates HCO3(-) and [5-13C]glutamate. These NMR data provide the first topographical map of the arrangement of substrates, metal ion activators, and allosteric modifiers on the Escherichia coli carbamoyl-phosphate synthetase dimer.
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PMID:A nuclear magnetic resonance study of the topography of binding sites of Escherichia coli carbamoyl-phosphate synthetase. 634 70

Fluorescence energy transfer experiments were used to measure distances between three fluorescently labeled sulfhydryl sites on Escherichia coli carbamoyl-phosphate synthetase, an unsymmetrical dimer. When five different combinations of fluorescent donor-acceptor pairs are used, the distance between site 1, located on the large subunit, and site 2, located on the small subunit, is in the range of 27-33 A. Similarly, the distance between site 1 and site 3 (large subunit) was approximately 27 A and between site 2 and site 3 was approximately 21 A. A similar approach was employed to determine distances between each sulfhydryl group and the ATP site(s), and in all cases no fluorescence quenching was observed using Cr3+ATP or Co(NH3)4ATP as substrate analogues. A lower limit could be calculated from these data, resulting in a distance of greater than or equal to 21 A from each sulfhydryl site to the ATP site. Additional experiments were performed to evaluate if the substrates ATP, HCO3(-), or glutamine or the allosteric modifiers ornithine, IMP, and UMP altered the distance relationships among the sulfhydryl sites. IMP and UMP produced a slight decrease in fluorescence between sites while glutamine and ATP produced a slight increase in fluorescence.
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PMID:Fluorescence energy transfer experiments with Escherichia coli carbamoyl-phosphate synthetase. 634 71

Some properties of carbamoyl-phosphate synthetase (ammonia) were studied in rat-liver mitochondria made selectively permeable by pretreatment with toluene. The Michaelis constants for NH3, MgATP and HCO-3 were 0.7, 1.2 and 2 mM respectively. N-Acetylglutamate activated the enzyme with a Ka of about 0.1 mM. At saturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 mM. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase required the presence of both free Mg2+ ions and MgATP, and was inhibited by Ca2+ ions and by N-carbamoylglutamate. The known activation of carbamoyl-phosphate synthetase by free Mg2+ is due to an increased affinity of the enzyme for N-acetylglutamate. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase was a slow process: at N-acetylglutamate concentrations below 0.5 mM maximal binding was not completed within 30 min. The rate of binding increased with increasing N-acetylglutamate concentrations. Dissociation of N-acetylglutamate from the enzyme was relatively fast, with a half-time of about 5 min. Under all conditions studied there was a close relationship between carbamoyl-phosphate synthetase activity and the amount of N-acetylglutamate bound to the enzyme. The data are discussed in relation to the control of carbamoyl-phosphate synthetase in the intact hepatocyte.
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PMID:Properties of carbamoyl-phosphate synthetase (ammonia) in rat-liver mitochondria made permeable with toluene. 688 64

1. When NH3 was added to isolated rat-liver mitochondria incubated with succinate and bicarbonate, oxidation of succinate was stimulated to a greater extent than could be accounted for by the net formation of carbamoyl phosphate. 2. Measurement of the rate of incorporation of [14C]bicarbonate into carbamoyl phosphate, after the mitochondria had been preincubated with NH3 and unlabelled bicarbonate, revealed that flux through carbamoyl-phosphate synthetase (ammonia) was much greater than the net formation of carbamoyl phosphate indicated. 3. It is concluded that part of the carbamoyl phosphate produced in the absence of ornithine is degraded. About 20% of the degradation can be accounted for by non-enzymatic reactions of carbamoyl phosphate outside the mitochondria. It is proposed that the remainder of the degradation of carbamoyl phosphate occurs by partial reversal of the reaction of carbamoyl-phosphate synthetase.
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PMID:Activity of carbamoyl-phosphate synthetase (ammonia) in isolated rat-liver mitochondria: cycling of carbamoyl phosphate in the absence of ornithine. 708 32

The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli has unveiled the existence of two molecular tunnels within the heterodimeric enzyme. These two interdomain tunnels connect the three distinct active sites within this remarkably complex protein and apparently function as conduits for the transport of unstable reaction intermediates between successive active sites. The operational significance of the ammonia tunnel for the migration of NH3 is supported experimentally by isotope competition and protein modification. The passage of carbamate through the carbamate tunnel has now been assessed by the insertion of site-directed structural blockages within this tunnel. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The structurally modified proteins G575L, A23L/G575S, and A23L/G575L exhibited a substantially reduced rate of carbamoyl phosphate synthesis, but the rate of ATP turnover and glutamine hydrolysis was not significantly altered. These data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain. The partial reactions of CPS have not been significantly impaired by these mutations, and thus, the catalytic machinery at the individual active sites has not been functionally perturbed.
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PMID:Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase. 1237 99

The Chinese fire-belly newt Cynops orientalis reverts to an aquatic mode of living when sexually mature. Despite living in water, sexually mature C. orientalis maintained high capacity for hepatic urea synthesis. However, it had a lower rate of urea production than other terrestrial amphibians because endogenous ammonia could diffuse out to the external medium as NH3. This conserves cellular energy because urea synthesis is energetically expensive. Simultaneously, C. orientalis also reduced the rate of urea excretion, and excreted 33% of the total nitrogenous waste as ammonia. Upon exposure to land, C. orientalis increased the rate of urea synthesis from accumulating endogenous ammonia. The increased rate of urea synthesis was within the inherent capacity of the hepatic ornithine-urea cycle; there was no induction of hepatic carbamoyl phosphate synthetase or ornithine transcarbamoylase activities and there was no reduction in ammonia production. When exposed to water containing 75 mmol.l(-1) NH4Cl, the rates of both urea synthesis and urea excretion increased. Under such experimental conditions, the ornithine-urea cycle may be operating close to its limit; glutamine began to accumulate in the body, and endogenous ammonia production via amino acid catabolism was reduced.
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PMID:Excretory nitrogen metabolism in the Chinese fire-belly newt Cynops orientalis in water, on land, or in high concentrations of environmental ammonia. 1461 Jun 82

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.
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PMID:African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia. 1509 44


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