Glutamine-dependent Carbamoyl-phosphate Synthetase Research Articles

CAD, A Multienzymatic Protein at the Head of de Novo Pyrimidine Biosynthesis.

CAD is a 1.5 MDa particle formed by hexameric association of a 250kDa protein that carries the enzymatic activities for the first three steps in the de novo biosynthesis of pyrimidine nucleotides: glutamine-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase and Dihydroorotase. This metabolic pathway is essential for cell growth and proliferation and is conserved in all living organisms. However, the fusion of the first three enzymatic activities of the pathway into a single multienzymatic protein only occurs in animals. In prokaryotes, by contrast, these activities are encoded as distinct monofunctional enzymes that function independently or by forming more or less transient complexes. Whereas the structural information about these enzymes in bacteria is abundant, the large size and instability of CAD has only allowed a fragmented characterization of its structure. Here we retrace some of the most significant efforts to decipher the architecture of CAD and to understand its catalytic and regulatory mechanisms.

Getting CAD in Shape: The Atomic Structure of Human Dihydroorotase Domain

CAD is a large multifunctional polypeptide that initiates and controls the de novo biosynthesis of pyrimidines in animals. In this issue of Structure, Grande-García and colleagues provide the first atomic information of this antitumoral target by reporting the crystal structure of the dihydroorotase domain of human CAD.

Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii

Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28 kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa ( Plasmodium, Babesia, Toxoplasma) or zoomastigina ( Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.

Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi

A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas’ disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6–5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5′-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6–5, pyr2, and pyr4 from the 5′ terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6–5, are covalently linked in the order opposite to mammalian pyr5–6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1–3–2, pyr4, and pyr5–6, for UMP synthesis. Peculiarities in thetrypanosomatid pyr6–5 gene product are discussed.

Quantification of PAP39 and PRS1 mRNA by competitive PCR

Mammalian glutamine-dependent carbamoyl phosphate synthetase (EC 2.7.2.9), the first enzyme of de novo pyrimidine nucleotide biosynthesis, was strongly inhibited by polyamines at concentrations of 10−4 to 10−3 M. Spermine was the most effective, followed in order by spermidine and putrescine. The inhibition was partially reversed by increasing the concentration of Mg2+ or MgATP2−, or by adding low concentrations of 5-phosphoribosyl 1-pyrophosphate, an allosteric activator of the enzyme. Polyamines increased the apparent Ka value of the enzyme for phosphoribosyl pyrophosphate. A possible physiological role of polyamines in widening the range of the effective concentrations of phosphoribosyl pyrophosphate as the activator for the enzyme is suggested.

Glutamine-dependent urea synthesis in elasmobranch fishes

Marine elasmobranchs (sharks, skates, and rays) synthesize and retain in their tissues high concentrations of urea (up to 0.4 M) and certain amines, such as trimethylamine oxide (up to 0.2 M), for the purpose of osmoregulation. Although the pathway of urea synthesis in ureosmotic elasmobranchs is similar to the classical urea cycle in ureotelic species, there are several properties that are unique. Indeed, this might be anticipated, since the primary function of urea synthesis in elasmobranchs is osmoregulation, rather than ammonia detoxification. The existence in different species of a major biosynthetic pathway resulting in the same end product, but which serves quite different physiological purposes in each species, can provide an opportunity for comparative investigations that contribute to our understanding of the regulatory properties and evolutionary development of the pathway. The purpose of this commentary is to consider the unique properties of urea synthesis in elasmobranchs and to comment on the comparative relationships to teleost fishes and mammalian species; most studies discussed here have been carried out with spiny dogfish shark, Squalus acanthias, a representative elasmobranch. Like ureotelic species, hepatic urea synthesis in ureosmotic elasmobranchs requires both mitochondrial and cytosolic enzymes, but there are notable differences (Fig. 1). The most significant difference is that the initial step of ammonia assimilation for urea synthesis in hepatic mitochondria of elasmobranchs is the formation of glutamine, which is subsequently utilized as the substrate for carbamoyl-phosphate synthesis (Anderson and Casey, J. Biol. Chem., 259, 456-462, 1984). This is due to the presence of high levels of glutamine synthetase (GSase) and a unique acetylglutamateand glutamine-dependent carbamoyl-phosphate synthetase (CPSase 111), both localized exclusively in the mitochondrial matrix (Casey and Anderson, J. Biol. Chem., 257,8449-8453, 1982, and Comp. Biochem. Physiol., 82B, 307-315,1985). GSase is a cytosolic enzyme in liver of mammalian species and in some, but not all, teleost fishes. In mammalian and amphibian species the first step of ammonia assimilation for urea synthesis in liver is direct conversion of ammonia to carbamoyl phosphate catalyzed by a corresponding mitochondrial acetylglutamateand ammoniadependent CPSase I (which cannot utilize glutamine as a substrate). The properties of elasmobranch mitochondrial CPSase I11 are similar to the mammalian mitochondrial CPSase I, except for the fact that glutamine rather than ammonia serves as the nitrogen-donating substrate (Anderson, J. Biol. Chem., 256, 12 228 12 238, 1981).

Mammalian carbamyl phosphate synthetase (CPS). DNA sequence and evolution of the CPS domain of the Syrian hamster multifunctional protein CAD.

Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish)

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.

Combined action of acivicin and d-galactosamine on pyrimidine nucleotide metabolism in hepatoma cells

The glutamine antagonist acivicin, l-(αS,5S)-α- amino-3- chloro-4,5- dihydro-5- isoxazoleacetic acid, strongly reduced CTP and GTP contents in AS-30D rat hepatoma cells in suspension. UTP only dropped to 63% of the respective control after 4hr; however, by combining acivicin with the uridylate-trapping sugar analogue d-galactosamine, a synergistic decrease in UTP contents to 7% of control was induced. Incorporation of 14CO 2 into purine and pyrimidine nucleotides followed by radio-high performance liquid chromatography showed marked inhibition of purine and pyrimidine biosynthesis de novo; the latter was reduced to 35% of control. The inhibitory potency of acivicin on glutamine-dependent carbamoyl-phosphate synthetase and consequently on de novo uracil nucleotide formation was also reflected by the complete suppression of the d-galactosamine-induced rise in total uridylate. Induction of UTP deficiency by interference with the first and rate-limiting step in pyrimidine biosynthesis de novo together with a trapping of uridylate by d-galactosamine may provide a promising approach to the chemotherapy of hepatocellular carcinoma.

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines.

All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively. Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ornithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction. The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoylphosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.

Assay of the Enzymes of Pyrimidine Synthesis by Thin Layer Ion Exchange Chromatography and Video-Densitometry (CV-Technique)

Abstract A simple and rapid method is described for the assay of the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase in minute amounts of liver tissue. From 60–120 μl assay mixtures 10–20 μl aliquots were chromatographed on thin layer ion exchange chromatoplates, and the amino acids were developed with ninhydrin. The intensity of the ninhydrin spots, i.e. the quantity of amino acids formed or consumed, was determined by video-densitometry and the specific activity of the enzymes was calculated.

Control of pyrimidine biosynthesis in the Ascaris ovary: regulatory properties of glutamine-dependent carbamoyl-phosphate synthetase and copurification of the enzyme with aspartate carbamoyltransferase and dihydroorotase.

Glutamine-dependent carbamoyl-phosphate synthetase, the first enzyme of the de novo biosynthetic pathway for pyrimidine nucleotides, was purified about twenty-fold from 105 000 x g supernatant of the Ascaris ovary homogenate. The enzyme activity was feedback-inhibited by UDP and UTP while it was stimulated by 5-phosphoribosyl 1-pyrophosphate. Most of the catalytic and regulatory properties of the Ascaris synthetase were similar to those of the mammalian synthetase. A significant difference is that the Ascaris enzyme was more strongly inhibited by UDP than by UTP whereas the mammalian enzyme is more sensitive to UTP than to UDP. The Ascaris enzyme was also inhibited by other various nucleoside diphosphates, such as dUDP, dADP and CDP, generally more strongly than by the corresponding nucleoside triphosphates. Aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway, were also demonstrated in the supernatant fraction. These two enzymes were copurified with the synthetase and the relative activities of the three enzymes remained nearly constant (1:850-890:50-60) throughout the purification. In a sucrose gradient centrifugation, the enzymes cosedimented as a single peak with a sedimentation coefficient (s20,w) of about 32 S under the condition used. These results strongly suggest that the enzymes exist as a multienzyme complex similar to those found in higher animals. The activity of the carbamoyltransferase was insensitive to nucleotides and related compounds. These results indicate that the synthetase plays a key role in the control of pyrimidine biosynthesis in the Ascaris ovary.

Glutamine-dependent carbamoyl-phosphate synthetase and control of pyrimidine biosynthesis in the parasitic helminth Schistosoma mansoni

1. 1. Carbomoyl-phosphate synthetase in Schistosoma mansoni utilizes l-glutamine as well as ammonia as nitrogen donor but does not require N- acetyl- l-glutamate for the activity. 2. 2. The enzyme activity was inhibited by UDP, UTP, ADP and AMP, among which UDP was the most effective. 3. 3. Aspartate carbamoyltransferase and dihydroorotase were also found and copurified with the synthetase. 4. 4. Relative activities among these three enzymes were 1:30–60:3–8 throughout the purification. 5. 5. These results suggest that the synthetase plays a key role in the control of pyrimidine biosynthesis de novo.

Bilateral enzymic changes in rat nodose ganglia following unilateral cervical vagotomy

Aspects of the metabolism of the nodose ganglia of the rat and their relationship to chromatolysis induced by unilateral cervical vagotomy were examined. The development of chromatolysis in the vagotomized ganglion followed a typical pattern. The changes in nucleic acid metabolism were elucidated by measurement of the activity of the pyrimidine biosynthesis specific enzyme: glutamine dependent carbamoyl phosphate synthetase. The activity of this enzyme showed a biphasic response supporting the conclusion that the first rapid increase in the rate of synthesis of ribosomal RNA may be a result of increased activity of the de novo pyrimidine biosynthetic pathway; a biphasic response also occurred in the enzyme activity in the contralateral ganglion. Lysosomal activity during the response was investigated by measuring the activity of acid phosphatase. There was also a bilateral change in the acid phosphatase activities of the two ganglia. This was more marked in the vagotomized ganglion, but followed a biphasic time response. The bilateral nature of the response observed in enzyme activities may reflect the similarities in metabolic requirements of axon regrowth in the vagotomized ganglion and axon collateral growth in the contralateral ganglion.

Multi-enzyme complex of glutamine-dependent carbamoyl-phosphate synthetase with aspartate carbamoyltransferase and dihydroorotase from rat ascites-hepatoma cells. Purification, molecular properties and limited proteolysis.

Glutamine‐dependent carbamoyl‐phosphate synthetase, the first enzyme of de novo pyrimidine nucleotide biosynthesis, was purified 1300‐fold from Yoshida ascites hepatoma cells (AH 13) as a multi‐enzyme complex with aspartate carbamoyltransferase and dihydroorotase, using dimethyl sulfoxide, glycerol and dithiothreitol as enzyme stabilizers. The ratios of the three enzyme activities were initially 1:65:9.2 and were essentially constant throughout the purification, and the final fraction had ratios of 1:37:11 (dihydroorotase assayed in the forward direction). The purified complex had a specific activity of the synthetase of 0.51 μmol × min−1× (mg protein)−1. It was essentially homogeneous on agarose/acrylamide composite gel electrophoresis, analytical ultra‐centrifugation, and electrofocusing; it had an apparent pI of 5.5. The complex had a sedimentation coefficient (s20, w) of 24.6 S in the presence of 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol, and its molecular weight was estimated to be 870000 by sedimentation equilibrium studies.On polyacrylamide gel electrophoresis in sodium dodecyl sulfate the complex migrated as a single protein band with a molecular weight of 210000, indicating that the complex is composed of four to five subunits of similar size. Sucrose gradient centrifugation in the presence of 1.5 M sodium chloride partially dissociated the three enzyme activities. Limited proteolysis of the complex with pancreatic elastase caused its dissociation and made separable the three activities. The synthetase was recovered as a 197000‐molecular‐weight fragment and the carbamoyltransferase as a 145000‐molecular‐weight fragment. Dihydroorotase was initially recovered as a 197000‐molecular‐weight fragment and was further converted to a 43000‐molecular‐weight fragment. It appears difficult to reconcile these results with the view that the three enzyme activities are located on the same polypeptide chain.

Glutamine-dependent carbamoyl phosphate synthetase: Polyamines inhibit the activity and modify the activating effect of 5-phosphoribosyl 1-pyrophosphate

Mammalian glutamine-dependent carbamoyl phosphate synthetase (EC 2.7.2.9), the first enzyme of de novo pyrimidine nucleotide biosynthesis, was strongly inhibited by polyamines at concentrations of 10 −4 to 10 −3 M. Spermine was the most effective, followed in order by spermidine and putrescine. The inhibition was partially reversed by increasing the concentration of Mg 2+ or MgATP 2−, or by adding low concentrations of 5-phosphoribosyl 1-pyrophosphate, an allosteric activator of the enzyme. Polyamines increased the apparent K a value of the enzyme for phosphoribosyl pyrophosphate. A possible physiological role of polyamines in widening the range of the effective concentrations of phosphoribosyl pyrophosphate as the activator for the enzyme is suggested.

Urea cycle enzymes in white blood cells—ii. Arginase activity in some types of human leukemia

1. 1. The activity of the “Krebs-Henseleit-Cycle” enzymes was investigated in crude extracts of leucocytes of patients with chronic lymphocytic leukemia (CLL), chronic granulocytic leukemia (CML) and osteomyelosclerosis (OMSL). 2. 2. The average arginase activity in 25 CLL leucocyte extracts is about 30 times lower than the value obtained previously from normal donors. In contrast, the arginase activities in 13 CML leucocyte extracts do not show significant differences to the extracts of normal donor leucocytes. 3. 3. No significant activities of N-acetylglutamate dependent carbamoylphosphate-synthetase (CPS I), glutamine dependent carbamoylphosphate-synthetase (CPS II), ornithine-carbamoyltransferase (OCT) and arginine-synthetase-system (ASS) were found.

Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver.

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.

Copurification of carbamoyl phosphate synthetase and aspartate transcarbamoylase from mouse spleen

A purification technique developed for the glutamine dependent carbamoyl phosphate synthetase from mouse spleen resulted in the copurification of aspartate transcarbamoylase. The two activities remained associated through (NH 4) 2SO 4 precipitation, hydroxylapatite adsorption and sucrose density gradient centrifugation. The pH curve of the aspartate transcarbamoylase exhibited two optima, one at pH 9.4 and a much higher one at pH 10.2. At pH 7.4 and 37°, aspartate transcarbamoylase had a K m for aspartate of 8.4 mM and V max of 0.35 μmoles product formed per min. per mg. protein. The K m for carbamoyl phosphate was 1.8 × 10 −3 mM. Activity of aspartate transcarbamoylase was not affected by any one of a number of pyrimidine nucleotides.