In Lactobacillus plantarum, carbamoyl phosphate is synthesized by two carbamoyl-phosphate synthetases (CPS): carbon dioxide differentiates the arginine-repressed from the pyrimidine-regulated CPS.
(9/122)
Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO(2). Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagoz et al., Gene 182:37-43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN). Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon. CP biosynthesis in L. plantarum was studied with three mutants (DeltaCPS-P, DeltaCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO(2)-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type L. plantarum without CO(2) enrichment may be due to the low affinity of CPS-A for its substrate CO(2) or to regulation of the CP pool by the cellular CO(2)/bicarbonate level. (+info)
Topology of binding sites for carbamyl phosphate in aspartate transcarbamylase from Escherichia coli. The use of pyridoxal phosphate as covalent probe.
(10/122)
Pyridoxal phosphate, a competitive inhibitor of aspartate transcarbamylase, binds to six sites in the catalytic and to twelve sites in the regulatory subunits of this hexameric protein. The properties of its association to the active sites of the enzyme are very similar to those observed with one of its substrates, carbamyl phosphate. It tightly binds to one half of the sites in the absence of succinate, an analogue of the second substrate. Since pyridoxal phosphate can be linked covalently to the protein by reduction, the distribution of the high affinity binding sites on catalytic trimers was studied after dissociation of modified holoenzyme. Electrophoresis of isolated subunits under non-denaturing conditions revealed four distinct bands, corresponding to trimers containing 0 to 3 pyridoxal phosphate derivatives. The distribution among the four species as a function of ligand concentration in the absence of succinate indicates that in the native oligomer, pyridoxal phosphate (and by extrapolation, carbamyl phosphate) binds to both catalytic trimers, rather than to three sites on a single subunit. (+info)
Effect of carbamoyl phosphate on nitrogenase in Anabaena cylindrica Lemm.
(11/122)
Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate. (+info)
Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases.
(12/122)
The iron of the binuclear active center of [NiFe]-hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity. Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the [NiFe] cluster contains a sequence motif characteristic of O-carbamoyl-transferases it was discovered that carbamoyl phosphate is essential for formation of active [NiFe]-hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands. A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated (+info)
Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes.
(13/122)
Two crystal structures of human ornithine transcarbamylase (OTCase) complexed with the substrate carbamoyl phosphate (CP) have been solved. One structure, whose crystals were prepared by substituting N-phosphonacetyl-L-ornithine (PALO) liganded crystals with CP, has been refined at 2.4 A (1 A=0.1 nm) resolution to a crystallographic R factor of 18.4%. The second structure, whose crystals were prepared by co-crystallization with CP, has been refined at 2.6 A resolution to a crystallographic R factor of 20.2%. These structures provide important new insights into substrate recognition and ligand-induced conformational changes. Comparison of these structures with the structures of OTCase complexed with the bisubstrate analogue PALO or CP and L-norvaline reveals that binding of the first substrate, CP, induces a global conformational change involving relative domain movement, whereas the binding of the second substrate brings the flexible SMG loop, which is equivalent to the 240s loop in aspartate transcarbamylase, into the active site. The model reveals structural features that define the substrate specificity of the enzyme and that regulate the order of binding and release of products. (+info)
Somatotropin-induced amino acid conservation in pigs involves differential regulation of liver and gut urea cycle enzyme activity.
(14/122)
Somatotropin (ST) treatment promotes animal growth and allows for the conservation of amino acids by increasing nitrogen retention and reducing ureagenesis and amino acid oxidation. To determine whether the improvement in amino acid conservation with ST treatment involves regulation of urea cycle enzyme activities in both liver and intestine, growing swine were treated with either ST (150 microg x kg(-1) x d(-1)) or saline for 7 d. Fully fed pigs (n = 20) were infused intravenously for 2 h with NaH(13)CO(3) followed by a 4-h intraduodenal infusion of [1-(13)C]phenylalanine. Arterial and portal venous blood and breath samples were obtained at baseline and steady-state conditions for measurement of amino acid and blood urea nitrogen (BUN) concentrations and whole-body phenylalanine oxidation. Urea cycle enzyme activities were determined in liver and jejunum. ST decreased BUN (-46%), arterial (-34%) and portal venous (-43%) amino acid concentrations and whole-body phenylalanine oxidation (-30%). The activities of carbamoylphosphate synthase-I (-45%), argininosuccinate synthase (-38%), argininosuccinate lyase (-23%), arginase (-27%), and glutaminase (-18%), but not of ornithine carbamoyltransferase, ornithine aminotransferase, or glutamate dehydrogenase were reduced in liver of ST-treated pigs. ST slightly increased intestinal activity of glutaminase (+9%) but did not affect that of any other enzymes. ST decreased hepatic, but increased jejunal, N-acetylglutamate (an essential allosteric activator of carbamoylphosphate synthase-I; -26% and +32%, respectively) and carbamoylphosphate (a substrate for ornithine carbamoyltransferase; -20% and +28%, respectively) content. These results demonstrate that the reduced amino acid catabolism with ST treatment in growing pigs involves a reduction in hepatic urea cycle enzyme activities. The effect of ST treatment on porcine urea cycle enzymes is tissue-specific and is associated with a reduction in substrate availability for hepatic ureagenesis. (+info)
Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus.
(15/122)
Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment. (+info)
HypF, a carbamoyl phosphate-converting enzyme involved in [NiFe] hydrogenase maturation.
(16/122)
HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active [NiFe] hydrogenases in Escherichia coli and other bacteria. To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity. The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors. As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate. In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate. This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate. Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate. The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand. (+info)