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)

It has been proposed that key enzymes of ureagenesis and the alanine aminotransferase activity predominate in periportal hepatocytes. However, ureagenesis from alanine, when measured in the perfused liver, did not show periportal predominance and even the release of the direct products of alanine transformation, lactate and pyruvate, was higher in perivenous cells. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea production from precursors such as lactate or pyruvate plus ammonia. In the present work these aspects were investigated in the bivascularly perfused rat liver. The results of the present study confirm that gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region. Alanine synthesis from lactate and pyruvate plus ammonia, however, predominated in the perivenous region. Furthermore, no predominance of ureagenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. These observations corroborate the view that data on enzyme activity or expression alone cannot be extrapolated unconditionally to the living cell. The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureagenesis in periportal cells. However, since urea synthesis occurs at high rates in all hepatocytes with the possible exclusion of those cells not possessing carbamoyl-phosphate synthase, it is probable that ureagenesis is equally important as an ammonia-detoxifying mechanism in the perivenous region.
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PMID:Flexibility of the hepatic zonation of carbon and nitrogen fluxes linked to lactate and pyruvate transformations in the presence of ammonia. 1769 Jan 75

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.
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PMID:Mutation analysis of carbamoyl phosphate synthetase: does the structurally conserved glutamine amidotransferase triad act as a functional dyad? 1845 50

The transfer of ammonia in carbamoyl phosphate synthetase (CPS) was investigated by molecular dynamics simulations and experimental characterization of mutations within the ammonia tunnel. In CPS, ammonia is derived from the hydrolysis of glutamine and this intermediate must travel approximately 45 A from the site of formation in the small subunit to the site of utilization in the large subunit. In this investigation, the migration of ammonia was analyzed from the exit of the small subunit through the large subunit where it ultimately reacts with the carboxy phosphate intermediate. Potential of mean force calculations along the transfer pathway for ammonia indicate a relatively low free-energy barrier for the translocation of ammonia. The highest barrier of 7.2 kcal/mol is found at a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. The environment of the ammonia tunnel from the exit of the small subunit to the turning gate in the tunnel is filled with clusters of water molecules and the ammonia is able to travel through this area easily. After ammonia passes through the turning gate, it enters a hydrophobic passage. A hydrogen bond then forms between the ammonia and Thr-249, which facilitates the delivery to a more hydrophilic environment near the active site for the reaction with the carboxy phosphate intermediate. The transport process from the turning gate to the end of the tunnel is favored by an overall downhill free-energy potential and no free-energy barrier higher than 3 kcal/mol. A conformational change of the turning gate, caused by formation of the carboxy phosphate intermediate, is consistent with a mechanism in which the reaction between ATP and bicarbonate triggers the transport of ammonia and consequently accelerates the rate of glutamine hydrolysis in the small subunit. A blockage in the turning gate passageway was introduced by the triple mutant C232V/A251V/A314V. This mutant is unable to synthesize carbamoyl phosphate using glutamine as a nitrogen source.
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PMID:A combined theoretical and experimental study of the ammonia tunnel in carbamoyl phosphate synthetase. 1956 82

The transport of carbamate through the large subunit of carbamoyl phosphate synthetase (CPS) from Escherichia coli was investigated by molecular dynamics and site-directed mutagenesis. Carbamate, the product of the reaction involving ATP, bicarbonate, and ammonia, must be delivered from the site of formation to the site of utilization by traveling nearly 40 A within the enzyme. Potentials of mean force (PMF) calculations along the entire tunnel for the translocation of carbamate indicate that the tunnel is composed of three continuous water pockets and two narrow connecting parts, near Ala-23 and Gly-575. The two narrow parts render two free energy barriers of 6.7 and 8.4 kcal/mol, respectively. Three water pockets were filled with about 21, 9, and 9 waters, respectively, and the corresponding relative free energies of carbamate residing in these free energy minima are 5.8, 0, and 1.6 kcal/mol, respectively. The release of phosphate into solution at the site for the formation of carbamate allows the side chain of Arg-306 to rotate toward Glu-25, Glu-383, and Glu-604. This rotation is virtually prohibited by a barrier of at least 23 kcal/mol when phosphate remains bound. This conformational change not only opens the entrance of the tunnel but also shields the charge-charge repulsion from the three glutamate residues when carbamate passes through the tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through the narrowest parts of the carbamate tunnel. The mutants retained only 1.7% and 3.8% of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild type CPS, respectively.
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PMID:Carbamate transport in carbamoyl phosphate synthetase: a theoretical and experimental investigation. 2018 43

A collection of Streptococcus sanguinis strains from patients with endocarditis (n = 21) and from the oral cavity (n = 34) was subjected to a multi-locus sequence typing analysis using seven housekeeping genes, carbamoyl-phosphate synthetase (carB), Co/Zn/Cd efflux system component (czcD), d-alanyl-d-alanine ligase (ddl), DNA polymerase III (dnaX), glucose-6-phosphate dehydrogenase (gdh), DNA-directed RNA polymerase, beta subunit (rpoB) and superoxide dismutase (sodA). The scheme was expanded by the inclusion of two the putative virulence genes, bacitracin-resistance protein (bacA) and saliva-binding protein (ssaB), to increase strain discrimination. Extensive intra-species recombination was apparent in all genes but inter-species recombination was also apparent with strains apparently harbouring gdh and ddl from unidentified sources and one isolate harboured a sodA allele apparently derived from Streptococcus oralis. The recombination/mutation ratio for the concatenated housekeeping gene sequences was 1.67 (95% confidence limits 1.25-2.72) and for the two virulence genes the r/m ratio was 3.99 (95% confidence limits 1.61-8.72); recombination was the major driver for genetic variation. All isolates were distinct and the endocarditis strains did not form distinct sub-clusters when the data were analysed using ClonalFrame. These data support the widely held opinion that infecting S. sanguinis strains are opportunistic human pathogens.
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PMID:Clonal structure of Streptococcus sanguinis strains isolated from endocarditis cases and the oral cavity. 2189 56

The lichen Peltigera aphthosa consists of a fungus and green alga (Coccomyxa) in the main thallus and of a Nostoc located in superficial packets, intermixed with fungus, called cephalodia. Dark nitrogenase activity (acetylene reduction) of lichen discs (of alga, fungus and Nostoc) and of excised cephalodia was sustained at higher rates and for longer than was the dark nitrogenase activity of the isolated Nostoc growing exponentially. Dark nitrogenase activity of the symbiotic Nostoc was supported by the catabolism of polyglucose accumulated in the ligh and which in darkness served to supply ATP and reductant. The decrease in glucose content of the cephalodia paralleled the decline in dark nitrogenase activity in the presence of CO2; in the absence of CO2 dark nitrogenase activity declined faster although the rate of glucose loss was similar in the presence and absence of CO2. Dark CO2 fixation, which after 30 min in darkness represented 17 and 20% of the light rates of discs and cephalodia, respectively, also facilitated dark nitrogenase activity. The isolated Nostoc, the Coccomyxa and the excised fungus all fixed CO2 in the dark; in the lichen most dark CO2 fixation was probably due to the fungus. Kinetic studies using discs or cephalodia showed highest initial incorporation of (14)CO2 in the dark in to oxaloacetate, aspartate, malate and fumarate; incorporation in to alanine and citrulline was low; incorporation in to sugar phosphates, phosphoglyceric acid and sugar alcohols was not significant. Substantial activities of the enzymes phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and carbamoyl-phosphate synthase (EC 2.7.2.5 and 2.7.2.9) were detected but the activities of PEP carboxykinase (EC 4.1.1.49) and PEP carboxyphosphotransferase (EC 4.1.1.38) were negligible. In the dark nitrogenase activity by the cephalodia, but not by the free-living Nostoc, declined more rapidly in the absence than in the presence of CO2 in the gas phase. Exogenous NH 4 (+) inhibited nitrogenase activity by cephalodia in the dark especially in the absence of CO2 but had no effect in the light. The overall data suggest that in the lichen dark CO2 fixation by the fungus may provide carbon skeletons which accept NH 4 (+) released by the cyanobacterium and that in the absence of CO2, NH 4 (+) directly, or indirectly via a mechanism which involves glutamine synthetase, inhibits nitrogenase activity.
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PMID:Nitrogenase activity and dark CO2 fixation in the lichen Peltigera aphthosa Willd. 2430 52

Effect of environmental hypertonicity, due to exposure to 300 mM mannitol solution for 7 days, on the induction of ureogenesis and also on amino acid metabolism was studied in the air-breathing walking catfish, C. batrachus, which is already known to have the capacity to face the problem of osmolarity stress in addition to other environmental stresses in its natural habitats. Exposure to hypertonic mannitol solution led to reduction of ammonia excretion rate by about 2-fold with a concomitant increase of urea-N excretion rate by about 2-fold. This was accompanied by significant increase in the levels of both ammonia and urea in different tissues and also in plasma. Further, the environmental hypertonicity also led to significant accumulation of different non-essential free amino acids (FAAs) and to some extent the essential FAAs, thereby causing a total increase of non-essential FAA pool by 2-3-fold and essential FAA pool by 1.5-2.0-fold in most of the tissues studied including the plasma. The activities of three ornithine-urea cycle (OUC) enzymes such as carbamoyl phosphate synthetase, argininosuccinate synthetase and argininosuccinate lyase in liver and kidney tissues, and four key amino acid metabolism-related enzymes such as glutamine synthetase, glutamate dehydrogenase (reductive amination), alanine aminotransaminase and aspartate aminotransaminase were also significantly up-regulated in different tissues of the fish while exposing to hypertonic environment. Thus, more accumulation and excretion of urea-N observed during hypertonic exposure were probably associated with the induction of ureogenesis through the induced OUC, and the increase of amino acid pool was probably mainly associated with the up-regulation of amino acid synthesizing machineries in this catfish in hypertonic environment. These might have helped the walking catfish in defending the osmotic stress and to acclimatize better under hypertonic environment, which is very much uncommon among freshwater teleosts.
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PMID:Influence of environmental hypertonicity on the induction of ureogenesis and amino acid metabolism in air-breathing walking catfish (Clarias batrachus, Bloch). 2505 41

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis.
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PMID:Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD. 3031 7


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