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Hexamita inflata can derive energy from the degradation of arginine via the arginine dihydrolase pathway. Carbamate kinase catalyses the third enzymatic step of the pathway synthesising ATP from the catabolism of carbamyl phosphate. This study reports the identification and characterisation of a carbamate kinase gene from this free-living diplomonad, together with measurements of carbamate kinase enzyme activity in cell-free extracts and a preliminary analysis of the carbamate kinase mRNA by reverse-transcription polymerase chain reaction. Analysis of the carbamate kinase gene revealed the use of non-canonical codons for glutamine. Phylogenetic studies showed a consistent close relationship between carbamate kinase sequences of H. inflata and Giardia intestinalis.

Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrirnidine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce ATP from ADP and CP. Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing link in the evolution of CP biosynthesis. The 1.5 Å resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open β-sheet core surrounded by α-helices, similar to those in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound ADP molecule, which does not dissociate at 22°C from the enzyme. The ADP nucleotide is sequestered in a ridge formed over. the C-edge of the core sheet, at the bottom of a large cavity, with the purine ring enclosed in a pocket specific for adenine. Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-like CPS is in fact a highly thermostable and very slow (at 37°C) CK that, in the extreme environment of P. furiosus, may have the new function of making, rather than using, CP. The thermostability of the enzyme may result from the extension of the hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair networks across several secondary structure elements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests that the slow rate at 37°C is possibly a consequence of slow product dissociation. (C) 2000 Academic Press., We thank the EU, ESRF and EMBL Grenoble for financial assistance and support for data collection. This work was supported by grants PM97-0134-C02-01 and PB95–0218 of the Dirección General de Enseñ anza Superior (DGES) of Spain. S.R.-M. is a predoctoral fellow of the Generalitat Valenciana and M.U. was a postdoctoral fellow of the Fundación Valenciana de Investigaciones Biomédicas-Bancaixa

15N isotope effects have been measured on the hydrolysis of glutamine catalyzed by carbamyl phosphate synthetase of Escherichia coli. The isotope effect in the amide nitrogen of glutamine is 1. 0217 at 37 degrees C with the wild-type enzyme in the presence of MgATP and HCO(3)(-) (overall reaction taking place). This V/K isotope effect indicates that breakdown of the tetrahedral intermediate formed with Cys 269 to release ammonia is the rate-limiting step in the hydrolysis. A full isotope effect of 1. 0215 is also seen in the partial reaction catalyzed by an E841K mutant enzyme, whose rate of glutamine hydrolysis is not affected by MgATP and HCO(3)(-). With wild-type enzyme in the absence of MgATP and HCO(3)(-), however, the (15)N isotope effect is reduced to 1. 0157. These isotope effects are interpreted in terms of partitioning of the tetrahedral intermediate whose rate of formation is dependent upon a conformation change which closes the active site after glutamine binding and prepares the enzyme for catalysis. An Ordered Uni Bi mechanism for glutamine hydrolysis that is consistent with the isotope effects and with the catalytic properties of the enzyme is proposed.

The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate- dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.

Aspartate transcarbamoylase (ATCase; EC 2.1.3.2 ) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase V max of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg 2+ , these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue N- phosphonacetyl- l -aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the Escherichia coli ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.

The Ura2 multidomain protein catalyses the first two steps of pyrimidines biosynthesis in Saccharomyces cerevisiae. It consists of a 240 kDa polypeptide which contains carbamyl phosphate synthetase and aspartate transcarbamylase domains. The Ura2 protein was believed to be nucleoplasmic, since one of the aspartate transcarbamylase reaction products, monophosphate, was reported to be precipitated by lead ions inside nuclei. However, this ultracytochemical approach was recently shown to give artifactual lead polyphosphate precipitates, and the use of cerium instead of lead failed to reveal this nucleoplasmic localization. Ura2 localization has therefore been undertaken by means of three alternative approaches based on the detection of the protein itself: (a) indirect immunofluorescence of yeast protoplasts; (b) immunogold labelling of ultrathin sections of embedded yeast cells (both approaches using affinity purified primary antibodies directed against the 240 kDa Ura2 polypeptide chain, or against a 22 residue peptide specific of the carbamyl phosphate synthetase domain); and (c) direct fluorescence of cells expressing an Ura2-green fluorescent protein hybrid. All three approaches localize the bulk of Ura2 to the cytoplasm, whereas the signals associated with the nucleus, mitochondria or vacuoles are close to or at the background level.

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit, and a small amidotransferase subunit, which belongs to the Triad family of glutamine amidotransferases. Previous studies have established that the reaction mechanism of the small subunit proceeds through the formation of a gamma-glutamyl thioester with Cys-269. The roles in the hydrolysis of glutamine played by the conserved residues, Glu-355, Ser-47, Lys-202, and Gln-273, were determined by mutagenesis. In the X-ray crystal structure of the H353N mutant, Ser-47 and Gln-273 interact with the gamma-glutamyl thioester intermediate [Thoden, J. B., Miran, S. G., Phillips, J. C., Howard, A. J., Raushel, F. M., and Holden, H. M. (1998) Biochemistry 37, 8825-8831]. The mutants E355D and E355A have elevated values of K(m) for glutamine, but the overall carbamoyl phosphate synthesis reaction is unperturbed. E355Q does not significantly affect the bicarbonate-dependent ATPase or glutaminase partial reactions. However, this mutation almost completely uncouples the two partial reactions such that no carbamoyl phosphate is produced. The partial recovery of carbamoyl phosphate synthesis activity in the double mutant E355Q/K202M argues that the loss of activity in E355Q is at least partly due to additional interactions between Gln-355 and Lys-202 in E355Q. The mutants S47A and Q273A have elevated K(m) values for glutamine while the V(max) values are comparable to that of the wild-type enzyme. It is concluded that contrary to the original proposal for the catalytic triad, Glu-355 is not an essential residue for catalysis. The results are consistent with Ser-47 and Gln-273 playing significant roles in the binding of glutamine.

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [14C]carbamoyl aspartate from [14C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b)is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu → Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.

The conflicting data on the binding of the two molecules of ATP that are involved in the overall reaction catalyzed by carbamoyl-phosphate synthetase (CPS) of Escherichia coli, and a mechanism recently proposed for this reaction, has led us to reexamine ATP binding using pulse/chase techniques. With [gamma-32P]ATP and bicarbonate in the pulse solution, there is a positive intercept at zero time of approximately 1 mol Pi/mol CPS in the plot of 32Pi formation against time, irrespective of whether the incubation is terminated by the addition of acid or by addition of a chase solution containing glutamine, excess unlabeled ATP and bicarbonate. The intercept is decreased to about 50% if the excess unlabeled ATP is added prior to the addition of the glutamine. These are the expected results if the intercept reflects the reversible formation of enzyme-bound ADP and carboxyphosphate. Approximately 0.6 mol carbamoyl [32P]phosphate/mol enzyme is formed in these experiments when the pulse step is terminated by addition to the chase solution. The ATP molecule that provides the phosphoryl group of carbamoyl phosphate, therefore, also binds to the enzyme in the absence of ammonia or glutamine and reacts in the chase to give carbamoyl phosphate before it can dissociate from the enzyme. At 1 mM ATP, the binding of both ATP molecules is essentially complete at 2.5 s, but the dissociation of the ATP that yields carbamoyl phosphate is extremely slow (t(1/2) of about 6 min at 22 degrees C; HCO3-, 40 mM), although it is faster in the absence of bicarbonate. The extreme sequestration from the aqueous environment of this ATP allows the enzyme-ATP complex to be separated from the surrounding ATP by centrifugal gel filtration. After two successive steps of gel filtration through Sephadex G-50 equilibrated with unlabeled ATP and bicarbonate, the majority of the radioactivity remaining in the solution is bound to the enzyme and is released as [gamma-32P]ATP if acid is added, or is converted to carbamoyl [32P]phosphate by addition to chase solution, without concomitant release of 32Pi. K+ is necessary in the pulse solution, but not in the chase solution, to demonstrate this binding. These findings and other confirmatory experiments demonstrate conclusively that, in the presence of K+, both ATP molecules bind to the enzyme in the absence of ammonia or glutamine. The bound ATP that yields Pi in the overall reaction is replaced relatively rapidly by exchange and by hydrolysis in the bicarbonate-dependent ATPase activity of the enzyme, whereas the bound ATP that provides the phosphoryl group of carbamoyl phosphate is replaced very slowly. The temporal pattern of carbamoyl [32P]phosphate formation from [gamma-32P]ATP, in pulse/chase experiments in which a small concentration of ammonia is added to the pulse solution, shows that, in the normal enzyme reaction, this last ATP molecule binds to the enzyme before ammonia. These findings exclude a recently proposed mechanism [Kothe, M., Eroglu, B., Mazza, H., Samudera, H. & Powers-Lee, S. (1997) Proc. Natl Acad. Sci. USA 94, 12348-12353] in which a single molecule of ATP bound at the catalytic center phosphorylates bicarbonate and provides the phosphoryl group of carbamoyl phosphate. A mechanism in which a single ATP molecule binds, followed by the binding of bicarbonate and ammonia (from glutamine) and the release of Pi before the second molecule of ATP is bound is also excluded. We have previously reported very similar findings for carbamoyl-phosphate synthetase (ammonia), strongly suggesting that the different types of CPS share a common mechanism. The virtual sequestration of the ATP that provides the phosphoryl group of carbamoyl phosphate is consistent with a palmate-binding site, with the nucleotide bound within a beta-sheet sandwich, and a loop closure mechanism triggered by the binding of bicarbonate or the formation of carboxyphosphate.

The insulin-like action of tungstate in diabetic rats (A. Barbera et al., 1994, J. Biol. Chem. 269, 20047–20053) prompted us to examine the effects of tungstate on the glucose-6-phosphatase system. Our results indicate that tungstate is a potent inhibitor of glucose-6-phosphatase, with a K i in the 10–25 μM range determined with native microsomes and in the 1–7 μM range determined with detergent-treated microsomes. With both preparations, simple linear competitive inhibition was observed versus glucose 6-phosphate (glucose-6-P) as substrate with the glucose-6-P phosphohydrolase activity of the enzyme. Tungstate was a simple linear competitive inhibitor versus carbamyl phosphate (carbamyl-P) and a linear noncompetitive inhibitor versus glucose with the carbamyl-P:glucose phosphotransferase activity of the glucose-6-phosphatase system. These findings, in addition to the observation that tungstate protected the enzyme against thermal inactivation, indicate that tungstate binds with high affinity and competes at the active site of the enzyme where the substrates glucose-6-P and carbamyl-P bind prior to catalysis. Our results suggest that potent inhibition of glucose-6-P hydrolysis by tungstate is likely responsible, at least in part, for the normalization of glycemia and the rebound in hepatic glucose-6-P levels observed in earlier studies in which tungstate exhibited insulin-like action in diabetic rats.

Most of the Indian air-breathing teleosts are primarily ammoniotelic, but appear to have retained the genes for the urea cycle enzymes, since a full complement of urea cycle enzymes have been reported for many of them. The ability to synthesize urea by these fish is probably due to their amphibious nature, and their normal habitat of swamps, where the water ammonia level may to be quite high, is uninhabitable to any typical freshwater teleosts. One of these air-breathing species, the singhi catfish ( Heteropneustes fossilis ), can tolerate very high ambient total ammonia concentrations (up to 75 mM ammonium chloride) for weeks without any deleterious effects. Transition from ammoniotelism to ureotelism occurs in some of these species of air-breathing fish when exposed to apparently stressful conditions such as higher ambient ammonia, to air, and also when they live in semidry condition inside mud during habitat drying. Although the real mechanism(s) of regulation of ureogenesis is not clear in these fish, given available data, it is hypothesized that the accumulation of ammonia within the body per se under the above stressful conditions is likely the internal modulator for enhanced ureogenesis mainly to avoid any build up of ammonia to a level that can be toxic to these fish. An active urea cycle is believed to predominate over uricolysis as a source of urea, even though both pathways are present in these air-breathing fish. The presence of significant levels of both carbamyl phosphate synthetase (CPS), CPS I-like and CPS III activities, reported in some air-breathing catfishes, may represent intermediate scenarios for a proposed evolutionary transition from CPS III to CPS I, or may play an important physiological adaptive role in the tolerance of these fish to high concentrations of ambient ammonia

Ornithine transcarbamylase (OTCase) catalyzes the reaction between l -ornithine and carbamyl phosphate in the first step of the urea cycle. 13 C isotope effects were measured in carbamyl phosphate, using OTCase obtained from E. coli in a one-column purification which yielded 30 mg of very pure enzyme from 5 l of cell culture. At near zero l -ornithine, the 13 C kinetic isotope effect was 1.0095, at high levels of l -ornithine (86 mM) the 13 C kinetic isotope effect was unity, and 0.83 mM ornithine was found to eliminate half the isotope effect. These results are indicative of an ordered kinetic mechanism in which carbamyl phosphate binds to the enzyme before l -ornithine. Similar experiments were performed using the slow substrate l -lysine in place of l -ornithine. At 90 mM l -lysine the 13 C kinetic isotope effect was large, 1.076. This value is most likely the intrinsic kinetic isotope effect with this substrate, and the chemistry of the enzyme catalyzed reaction has become rate limiting.

The formation of carbamoyl phosphate is catalyzed by a single enzyme using glutamine, bicarbonate and two molecules of ATP via a reaction mechanism that requires a minimum of four consecutive reactions and three unstable intermediates. The recently determined X-ray crystal structure of carbamoyl phosphate synthetase has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein. It has been demonstrated that the amidotransferase domain within the small subunit of the enzyme from Escherichia coli hydrolyzes glutamine to ammonia via a thioester intermediate with Cys269. The ammonia migrates through the interior of the protein, where it reacts with carboxy phosphate to produce the carbamate intermediate. The carboxy phosphate intermediate is formed by the phosphorylation of bicarbonate by ATP at a site contained within the amino-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the carboxy-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate. The entire journey from substrate to product covers a distance of nearly 100 A.

UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.

The kinetic and regulatory properties of aspartate carbamoyltransferase (ACTase) of the human pathogen Helicobacter pylori were studied in situ in cell-free extracts. The presence of enzyme activity was established by identifying the end product as carbamoylaspartate using nuclear magnetic resonance spectroscopy. Activity was measured in all strains studied, including recent clinical isolates. Substrate saturation curves determined employing radioactive tracer analysis or a microtiter colorimetric assay were hyperbolic for both carbamoyl phosphate and aspartate, and there was no evidence for substrate inhibition at higher concentrations of either substrate. The apparent K m were 0.6 and 11.6 m m for carbamoyl phosphate and aspartate, respectively. Optimal pH and temperature were determined as 8.0 and 45°C. Activity was observed with the l - but not the d -isomer of aspartate. Succinate and maleate inhibited enzyme activity competitively with respect to aspartate. The carbamoyl phosphate analogues acetyl phosphate and phosphonoacetic acid inhibited activity in a competitive manner with respect to carbamoyl phosphate. With limiting carbamoyl phosphate purine and pyrimidine nucleotides, tripolyphosphate, pyrophosphate, and orthophosphate inhibited competitively at millimolar concentrations. Ribose and ribose 5-phosphate at 10 m m concentration showed 20 and 35% inhibition of enzyme activity, respectively. N -Phosphonoacetyl- l -aspartate (PALA) was the most potent inhibitor studied, with 50% inhibition of enzyme activity observed at 0.1 μ m concentration. Inhibition by PALA was competitive with carbamoyl phosphate ( K i = 0.245 μ m ) and noncompetitive with aspartate. The kinetic and regulatory data on the activity of the H. pylori enzyme suggest it is a Class A ACTase, but with some interesting characteristics distinct from this class.

The primary mechanisms proposed for acetaminophen-induced hepatic necrosis should deplete protein thiols, either by covalent binding and thioether formation or by oxidative reactions such as S-thiolations. However, in previous studies we did not detect significant losses of protein thiol contents in response to administration of hepatotoxic doses of acetaminophen in vivo. In the present study we employed derivatization with the thiol-specific agent monobromobimane and separation of proteins by SDS–PAGE to investigate the possible loss of specific protein thiols during the course of acetaminophen-induced hepatic necrosis. Fasted adult male mice were given acetaminophen, and protein thiol status was examined subsequently in subcellular fractions isolated by differential centrifugation. No decreases in protein thiol contents were indicated, with the exception of a marked decrease in the fluorescent intensity, but not of protein content, as indicated by staining with Coomassie blue, of a single band of approximately 130 kDa in the mitochondrial fractions of acetaminophen-treated mice. This protein was identified by isolation and N-terminal sequence analysis as carbamyl phosphate synthetase-I (CPS-I) (EC 6.3.4.16). Hepatic CPS-I activities were decreased in mice given hepatotoxic doses of acetaminophen. In addition, hepatic glutamine synthetase activities were lower, and plasma ammonia levels were elevated in mice given hepatotoxic doses of acetaminophen. The observed hyperammonemia may contribute to the adverse effects of toxic doses of acetaminophen, and elucidation of the specific mechanisms responsible for the hyperammonemia may prove to be useful clinically. However, the preferential depletion of protein thiol content of a mitochondrial protein by chemically reactive metabolites generated in the endoplasmic reticulum presents a challenging and potentially informative mechanistic question.

The allosteric enzyme aspartate transcarbamylase from Escherichia coli (ATCase) displays regulatory properties that involve various conformational changes, including a large quaternary structure rearrangement. This entails a major change in its solution X-ray scattering curve upon binding substrate analogues, thereby providing a direct means to monitor the amount of different quaternary structures present in solution. Scattering curves in the presence of variable concentrations of several such substrate analogues were recorded using an area detector. Data were analyzed by singular-value decomposition without any prior assumption as to the number of quaternary structure states. They can all be accounted for with only two states. The structural transition appears to be concerted, a conclusion whose validity has been extended to higher resolution and to other combinations of ligands than previously studied using a linear detector. The titration curve with the bisubstrate analogue N-(phosphonacetyl)-l-aspartate (PALA) alone is identical to that previously obtained, while that in the additional presence of carbamyl phosphate (CP) shows a clear R shift. However, the scattering pattern of the ATCase–CP complex is shown to be different from the T pattern of the unligated enzyme, though not a linear combination of the Tand R patterns. Therefore, we conclude that CP acts by modifying the quaternary structure of the unligated enzyme to a T-like structure, a structure more easily converted to the R conformation by subsequent addition of PALA.

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.

Carbamoyl-phosphate synthetases (CPSases) utilize two molecules of ATP at two homologous domains, B and C, with ATP(B) used to form the enzyme-bound intermediate carboxy-phosphate and ATP(C) used to phosphorylate the carbamate intermediate. To further define the role of one CPSase peptide suggested by affinity labeling studies to be near the ATP(B) site, we have carried out site-directed mutagenic analysis of peptide 234-242 of the Saccharomyces cerevisiae arginine-specific CPSase. Mutants E234A, E234D, E236A, E236D and E238A were unable to complement the CPSase-deficient yeast strain LPL26 whereas mutants Y237A, E238D, R241K, R241E and R241P supported LPL26 growth as well as wild-type CPSase. Kinetic analysis of E234A and Y237A indicated impaired utilization of ATP(B) but not of ATP(C). D242A, a temperature-sensitive mutant, retained no detectable activity when assayed in vitro. These findings, together with the affinity labeling data and primary sequence analysis, strongly suggest that the yeast CPSase peptide 234-242 is located at the ATP(B) site and that some of its residues are important for functioning of the enzyme. D242 appears to occupy a critical structural position and E234, E236 and E238 appear to be critical for function, with the spatial arrangement of the carboxyl side chain also critical for E234 and E236.

Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide. In cells from exponential batch cultures, deposits of cerium phosphate delineated simultaneously or alternatively membranes of the secretory pathway: nuclear envelope, endoplasmic reticulum, Golgi complex and the plasmalemma; mitochondrial membranes and intramitochondrial fibrous component were labelled as well. Deposits of cerium phosphate were never observed in the nucleoplasm. Cells incubated in the absence of cerium or ATCase substrates and mutant S. cerevisiae cells lacking ATCase activity served as controls. Small round electron-dense condensates were found to be randomly distributed within some cells, both in control and experimental runs, in the nucleoplasm, cytoplasm and mitochondrial matrix and represented undefined osmicated endogenous compounds. Our results suggest that the synthesis of pyrimidine precursors occurs in membranes, where compounds such as UDP-glucose and CDP-diglycerides are needed for membrane and/or yeast cell wall synthesis. The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified.

A new continuous coupled uv-spectrophotometric assay is described for two phosphate-releasing enzymes, aspartate transcarbamylase and ATPase of herpes simplex virus (HSV). Phosphate release is coupled to the phosphorolysis of the nucleoside analog 7-methylinosine (m7Ino) catalyzed by purine nucleoside phosphorylase. When this reaction is monitored at 291 nm, the coupled assay can readily detect 10 nmol Pi released/min. Our method offers advantages over a recently reported continuous assay devised for measuring aspartate transcarbamylase activity using the nucleoside analog methylthioguanosine (MESG) as the linking substrate. In contrast to MESG, m7Ino is easily and inexpensively synthesized and is also commercially available. The spectrophotometric signal at 291 nm, produced by the difference in the extinction coefficients between nucleoside substrate and the base product, is significant over a much wider pH range than the signal difference between MESG and its phosphorolysis product at 360 nm. Saturation curves for aspartate and carbamyl phosphate and pH rate profiles have been reproduced using the purine nucleoside phosphorylase/m7Ino coupled assay. Initial velocity patterns constructed over micromolar to millimolar concentrations of aspartate and carbamyl phosphate yielded four kinetic parameters simultaneously. To further illustrate the application of this coupled assay, kinetic parameters were determined for the DNA-dependent ATPase reaction of HSV helicase-primase.

Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation.

The Indian air-breathing teleost fish Heteropneustes fossilis has been shown to have a functional urea cycle and to be able to switch from ammoniotelic to ureotelic nitrogen metabolism when exposed to high levels of ammonia or air. The objective of this study was to identify the type of carbamyl phosphate synthetase (CPS) catalyzing the first step of the urea cycle in H. fossilis . Mitochondrial CPS III [glutamine- and N-acetyl-L-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellar localization were found in kidney. Unexpectedly, a CPS I-like activity (ammonia- and NAG-dependent) was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney. The urea cycle-related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle-related CPS I in ureotelic mammalian and amphibian species. Whether or not this CPS I-like activity 1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or 2) represents an adapted CPS III activity in H. fossilis , these results suggest that the presence of both CPS I-like and CPS III activities may play an important physiological adaptive role in the tolerance of these fish to high concentrations of external ammonia.

It is generally known that carbamyl phosphate, a precursor of nucleotides, spontaneously degrades to cyanate in organisms. In the present study, we revealed the presence of an alkaline-labile cyanate-derivative in Solanum tuberosum L., which released cyanate in alkaline solution, and elucidated that it was a reaction product of cyanate with dehydroascorbate. The reaction mechanism was revealed as follows : dehydroascorbate existing as a hemiacetal form in neutral aolution reacts with cyanate to produce ultimately a spiro-compound consisted of hemiacetal and oxazolidone rings. The structure of the end product was identified by spectroscopic and X-ray analyses using a synthetic compound. This fact demonstrates that this reaction is a novel metabolic pathway of endogenous cyanate in organisms.

Urea cycle disorders are relatively rare but well-established causes of postpartum coma and death. Such clinical presentations have been reported previously in ornithine transcarbamylase and carbamyl phosphate synthetase deficiencies.We describe a woman, without prior symptoms of metabolic disease, who presented with hyperammonemia and psychiatric symptoms in the postpartum period. Initial diagnoses included acute fatty liver of pregnancy and postpartum psychosis. She was later found to have argininosuccinate synthetase deficiency after further metabolic investigations. Rare heterozygous mutations in the argininosuccinate synthetase gene were identified.Urea cycle disorders may present initially with postpartum psychiatric symptoms and may represent an underrecognized cause of "postpartum psychosis." We recommend obtaining metabolic studies in women with neurologic or severe psychiatric symptoms in the postpartum period.

In order to detoxify ammonia, mammalian livers use carbamoyl phosphate synthetase (CPS) and glutamine synthetase (GS) for conversion into respective non-toxic urea and glutamine. CPS is expressed in the periportal hepatocytes whereas GS is contained in the pericentral hepatocytes.To examine the expressional changes of CPS and GS in the liver being induced to become cirrhotic by hepatotoxin thioacetamide (TAA).Twenty-five male Wistar rats were divided into 5 groups of 5 animals each. Group 1 was for control. Groups 2 to 5 were treated with 200 mg/kg TAA intraperitoneally three times weekly for 1, 2, 3 and 4 months respectively. The immunohistochemical technique was employed in order to elucidate the expression of CPS and GS in each animal group.As centro-central fibrous bridging developed in the course of TAA treatment, expression of CPS declined dramatically and that of GS was no longer restricted to the pericentral hepatocytes. In month 4, CPS-positive hepatocytes were only found in some regenerative nodules, whereas GS expression became confined to the nodular periphery. Proper CPS staining required tissue fixation in a mixture of methanol, acetone and water (2:2:1 v/v) as opposed to 4% paraformaldehyde.In response to the hepatotoxin TAA, the liver attempts to regenerate by means of conserving persistent CPS-positive hepatocytes and rearranging GS-positive hepatocytes in response to vascular obstruction.

N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.

The presence and activities of the enzymes of the urea cycle in the bacterium Helicobacter pylori were investigated employing one- and two-dimensional NMR spectroscopy and radioactive tracer analysis. Cell suspensions, lysates and membrane preparations generated L-ornithine and ammonium at high rates in incubations with L-arginine, indicating the presence of arginase activity. Anabolic ornithine transcarbamoylase (OTCase) activity was identified by the formation of heat-stable products in incubations of cell-free extracts with ornithine and radiolabelled carbamoyl phosphate. The heat-labile product that resulted from incubations of cell-free extracts with citrulline radiolabelled in the guanidino moiety revealed the presence of catabolic OTCase activity. Argininosuccinate formation and catalysis indicated the presence of argininosuccinate synthetase and argininosuccinase activities. The findings suggested that H. pylori has a urea cycle which acts as an effective mechanism to extrude excess nitrogen from cells.

Treatment of ornithine carbamoyltransferase from dolphin Stenella with pyridoxal phosphate, followed by reduction with NaBH4 resulted in complete loss of enzyme activity. The phosphate alone or the substrate analogue 2-aminovaleric acid moderately decreased the extent of inactivation, while carbamoyl phosphate plus 2-aminovaleric acid provided complete protection from inactivation. The partially inactivated enzyme showed K(m) values for substrates equivalent to those of native enzyme and lowered Kcat values. Two lysyl residues were substantially modified in the absence of ligands but only one of them was responsible for the inactivation of catalytic activity. Modification of a single subunit was sufficient to completely abolish the catalytic activity of the trimeric enzyme. The lysine involved has been identified as lysine 56 on the known primary structure of homologous human liver enzyme.

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.

A mutant (M45) of the cyanobacterium Synechococcus sp. strain PCC 7942, which is defective in active transport of nitrate, was used for the studies of the nitrogen regulation of the genes involved in nitrate and CO2 assimilation. In a medium containing 30 mM nitrate as the nitrogen source, M45 grew under constant stress of nitrogen deficiency and accumulated a five-times-larger amount of the transcript of nirA, the gene for nitrite reductase, compared with nitrate-grown wild-type cells. By contrast, the level of the transcript of rbcL, the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was 40% of the wild-type level. Addition of ammonium to the culture of M45 abolished the accumulation of the nirA transcript and stimulated the accumulation of the rbcL transcript, showing that ammonium repressed and activated the transcription of nirA and rbcL, respectively. Glutamine, the initial product of ammonium fixation, also showed negative and positive effects on nirA and rbcL, respectively. One of the metabolites of glutamine, carbamoylphosphate, and its decomposition product, cyanate, were found to repress nirA and also to markedly activate rbcL. Cyanate negatively regulated another ammonium-repressible gene, glnA, but had no effect on the psbAI and rps1 genes. The effects of cyanate were not ascribable to the ammonium and CO, resulting from its decomposition. These findings suggested that cyanate may act as a regulator of the ammonium-responsive genes involved in carbon and nitrogen assimilation in the cyanobacterium.

Dietary calorie restriction (CR) delays age-related physiologic changes, increases maximum life span, and reduces cancer incidence. Here, we present the novel finding that chronic reduction of dietary calories by 50% without changing the intake of dietary protein induced the activity of mouse hepatic carbamyl phosphate synthetase I (CpsI) 5-fold. In liver, CpsI protein, mRNA, and gene transcription were each stimulated by approximately 3-fold. Thus, CR increased both the rate of gene transcription and the specific activity of the enzyme. Short-term feeding studies demonstrated that higher cpsI expression was due to CR and not consumption of more dietary protein. Intestinal CpsI activity was stimulated 2-fold, while its mRNA level did not change, suggesting enzyme activity or translation efficiency was stimulated. CpsI catalyzes the conversion of metabolic ammonia to carbamyl phosphate, the rate-limiting step in urea biosynthesis. cpsI induction suggests there is a shift in the metabolism of calorie-restricted animals toward protein catabolism. CpsI induction likely facilitates metabolic detoxification of ammonia, a strong neurotoxin. Enhanced protein turnover and metabolic detoxification may extend life span. Physiologic similarities between calorie-restricted and hibernating animals suggest the effects of CR may be part of a spectrum of adaptive responses that include hibernation.

Urea synthesis from ammonia, glutamine and arginine was determined in enterocytes from newborn (0-day-old), 2-21-day-old suckling, and 29-58-day-old post-weaning pigs. Pigs were weaned at 21 days of age. Cells were incubated for 30 min at 37 degrees C in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing (i) 0.5-2 mM NH4Cl plus 0.05-2 mM ornithine and 2 mM aspartate, (ii) 1-5 mM glutamine, or (iii) 0.5-2 mM arginine. In enterocytes from newborn and suckling pigs, there was no measurable synthesis of urea from ammonia, glutamine or arginine, and analysis of amino acids by a sensitive fluorimetric HPLC method revealed the formation of negligible amounts of ornithine from arginine. In contrast, in cells from post-weaning pigs, relatively large amounts of urea and ornithine were produced from ammonia, glutamine and arginine in a dose-dependent manner. To elucidate the mechanism of the developmental change of urea synthesis in pig enterocytes, the activities of urea-cycle enzymes were determined. The activities of enterocyte carbamoyl phosphate synthase I and ornithine carbamoyltransferase were lower in post-weaning pigs than in suckling ones, whereas there was no difference in arginino-succinate lyase. The activities of argininosuccinate synthase and arginase were increased by 4-fold and 50-100-fold, respectively, in enterocytes from post-weaning pigs compared with suckling pigs. The induction of arginase appears to be sufficient to account for the formation of urea from ammonia, glutamine and arginine in post-weaning pig enterocytes. These results demonstrate for the first time the presence of synthesis of urea from extracellular or intramitochondrially generated ammonia in enterocytes of post-weaning pigs. This hitherto unrecognized urea synthesis in these cells may be a first line of defence against the potential toxicity of ammonia produced by the extensive intestinal degradation of glutamine (a major fuel for enterocytes) and derived from diet and luminal micro-organisms.

Enzymatic synthesis of carbamoyl phosphate Carbamoyl phosphate is synthesized irreversibly by carbamoyl-phosphate synthase (CPS; EC6.3.4.16 and EC 6.3.5.5) in the first step of the routes of biosynthesis of pyrimidines, arginine and urea. In most bacteria, including Escherichia cofi, the same CPS is used for arginine and pyrimidine synthesis, whereas in higher organisms different CPS enzymes are used for making pyrimidines and argininelurea. Carbamoyl phosphate is also synthesized by carbamate kinase, an enzyme found in some bacteria that grow anaerobically using arginine as the major nutrient. Carbamate kinase uses ATP to phosphorylate carbamate reversibly, but its physiological role is to generate ATP from ADP and carbamoyl phosphate. The carbamoyl phosphate used in this reaction is generated from citrulline and phosphate by catabolic ornithine transcarbamylase. The reactions catalysed by carbamate kinase ( 1 ) and CPS (2) are compared below

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO-) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO- and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5'-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.

Summary ArcA is one of several transcription factors required for optimal anaerobic induction of the pyruvate formatelyase (pfl) operon. To aid the study at the molecular level of the interaction of ArcA with the pfl promoter-regulatory region we developed a procedure for the isolation of ArcA. The purification of ArcA involved chromatography in heparin agarose, hydroxylapatite and Mono-Q matrices and delivered a protein that was >95% pure. Gel retardation assays demonstrated that ArcA bound specifically to the pfl regulatory region. Three distinct ArcA–DNA complexes could be resolved depending on the ArcA concentration used. This finding suggested that either multiple ArcA-binding sites are present in the regulatory region or that ArcA can oligomerize at one or more sites. The DNA-binding activity of ArcA could be increased an estimated 10-fold by prior incubation of the protein with carbamoyl phosphate, suggesting that phosphorylation activates DNA binding or oligomerisation. DNase I footprint analyses identified four sites that were protected by ArcA from cleavage. Two of these sites spanned the transcription start site and —10 regions of promoters 6 and 7, while a third site partially overlapped the characterized binding site of integration host factor (IHF). ArcA exhibited the highest affinity for a stretch of DNA located between the IHF site and the transcription start site of promoter 7. These results are congruent with the hypothesis that a higher-order nucleoprotein complex comprising several proteins, including ArcA, is required to activate transcription from the multiple promoters of the pfl operon.

The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases. The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified. The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active. The k is 347-fold higher and the K is 40-fold lower than the complete GLNase domain. Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity. The 19-kDa amino half, the interaction subdomain, binds tightly to the E. coli CPSase large subunit. Thus, the GLNase domain consists of two subdomains which can autonomously fold and function. The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis. The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.

We report the effect of the ornithine transcarbamylase (OTC) transgene composed of 1.3 kb of the 5' flanking region of the rat OTC gene fused to rat OTC cDNA on urinary orotic acid excretion in OTC-deficient spf-ash (sparse-fur with abnormal skin and hair) mice during overnight-starvation and nitrogen loading. During starvation, spf-ash mice with about 6% and 2% of control levels of OTC activity in the liver and small intestine excreted a large amount of orotic acid in the urine. Transgenic spf-ash mice with about 10% and 30% of the control OTC activities in the liver and small intestine did not excrete more than the normal level of orotic acid. Accidental parasitization of transgenic spf-ash mice with ticks (Myocoptes musculinus) resulted in decrease of the OTC activities in the liver and small intestine to the levels in spf-ash mice, and increased excretion of orotic acid. During extermination of the ticks, the mice showed varied levels of OTC activity and orotic acid excretion. On nitrogen loading, transgenic spf-ash mice as well as spf-ash mice excreted larger amounts of orotic acid, while control mice showed no increase in its excretion. The levels of urinary orotic acid were inversely correlated to the logarithms of the OTC activities in the liver and small intestine, the correlation being significantly higher with intestinal OTC than with hepatic OTC activity. These results suggest that the level of OTC activity in the small intestine is important for production of orotic acid.

Bacillus subtilis No. 428 is a cytidine-producing mutant strain derived from wild-type strain No. 122. To identify the rate-determining factor for production of cytidine by strain No. 428, we examined the properties of the strain. We found that carbamyl phosphate synthetase P (CPSase P) from strain No. 428 was sensitive to feedback inhibition by uridine 5′-monophosphate (UMP), and therefore, this enzyme reaction was the rate-determining step for cytidine production. Thus, we introduced a mutation causing lack of feedback inhibition of CPSase P into the CPSase P-deficient mutant of strain No. 428 by homologous recombination. Some of the transformants had higher productivity of cytidine. Among them, strain No. 515 produced 18.8 mg/ml cytidine in the culture. CPSase P from strain No. 515 was freed from feedback inhibition by UMP, having obtained the characteristic of the donor.

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions ofde novopyrimidine biosynthesis: glutamine-dependentcarbamyl phosphate synthetase,aspartate transcarbamylase anddihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Å resolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space groupC2221, with unit-cell parametersa= 82.1,b= 159.3,c= 61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.

The genome of the major intestinal archaeon Methanobrevibacter smithii contains a complex gene system coding for carbamoyl phosphate synthetase (CPSase) composed of both full-length and reduced-size synthetase subunits. These ammonia-metabolizing enzymes could play a key role in controlling ammonia assimilation in M. smithii, affecting the metabolism of gut bacterial microbiota, with an impact on host obesity. In this study, we isolated and characterized the small (41 kDa) CPSase homolog from M. smithii. The gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was purified in one step. Chemical cross-linking and size exclusion chromatography indicated a homodimeric/tetrameric structure, in accordance with a dimer-based CPSase activity and reaction mechanism. This small enzyme, MS-s, synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia and catalyzed the same ATP-dependent partial reactions observed for full-length CPSases. Steady-state kinetics revealed a high apparent affinity for ATP and ammonia. Sequence comparisons, molecular modeling, and kinetic studies suggest that this enzyme corresponds to one of the two synthetase domains of the full-length CPSase that catalyze the ATP-dependent phosphorylations involved in the three-step synthesis of carbamoyl phosphate. This protein represents the smallest naturally occurring active CPSase characterized thus far. The small M. smithii CPSase appears to be specialized for carbamoyl phosphate metabolism in methanogens.

Glu-86, which interacts with the side chain of Arg-54 across the C1-C2 interface of Escherichia coli aspartate transcarbamoylase, tethers the end of the flexible 80's loop, which moves into the active site during the T to R transition. In order to determine whether this interaction is important for the correct positioning of the 80's loop and Arg-54 at the active site and also for the structural stabilization of the enzyme, a mutant version was created in which Glu-86 was replaced by Gln (Glu-86-->Gln). Although the mutant holoenzyme exhibits almost normal homotropic cooperativity, both the holoenzyme and catalytic subunit exhibit substantial reductions in activity and affinity for aspartate and carbamyl phosphate. Furthermore, the mutant holoenzyme shows a marked decrease in the activation by ATP and by the bisubstrate analog N-(phosphonoacetyl)-L-aspartate, reduced inhibition by CTP, as well as reduced affinities for these ligands. Results from molecular dynamics simulations of the Glu-86-->Gln and Glu-86-->Ala enzymes suggest that the positions of the 80's loop and Arg-54 are significantly perturbed by the introduction of these mutations. Taken together, these results indicate that the interaction between Glu-86 and Arg-54 is important for the formation of the high affinity, high activity form of the enzyme by stabilizing the correct position of the 80's loop and Arg-54 at the active site. Heat inactivation experiments also demonstrated that Glu-86 plays a significant role in the structural stabilization of the C1-C2 interface, since the temperature required for loss of half of the activity of the Glu-86-->Gln catalytic subunit is reduced by 5 degrees C relative to the wild-type catalytic subunit.

The amino acid sequence of carbamyl phosphate synthetase (CPS) III from liver of spiny dogfish shark Squalus acanthias was deduced from the nucleotide sequence of its cDNA. Alignment of the derived amino acid sequence of CPS III with sequences of rat and frog CPS I and hamster CPS II reveals a high degree of amino acid identity, indicating that CPS III shares the same common ancestral genes as CPSs I and II. All of the CPSs examined show a high conservation of sequences in the adenine nucleotide binding domains and in residues that have been implicated in catalysis. The active-site cysteine residue required for glutamine-dependent activity by CPS II is preserved in the sequence of CPS III. Nevertheless, analysis of the protein sequences indicates that CPS III is more closely related to CPS I than to CPS II. The structure of CPS III, which is composed of a single polypeptide, is consistent with the view that CPS III evolved by fusion of separate genes coding for the glutaminase and synthetase domains of the enzyme and, like other CPSs, the synthetase domain evolved by duplication and fusion of an ancestral kinase gene. These results, together with the recent finding that frog CPS I retains the active site cysteine residue in the glutaminase domain required for glutamine-dependent activity, indicate that other amino acid substitutions critical for glutamine-dependent activity preceded loss of this catalytic cysteine residue. The results described here together with earlier biochemical evidence support the view that acetylglutamate and glutamine-dependent CPS III found in invertebrates and fish species represents an intermediate in the evolution of ancestral glutamine-dependent CPS II toward the acetylglutamate and ammonia-dependent CPS I of ureotelic terrestrial vertebrates.