Transcarbamoylase Activity Research Articles

Metabolic resistance: the protection of enzymes against drugs which are tight-binding inhibitors by the accumulation of substrate.

Blockade of a metabolic pathway by interaction of a drug with a particular 'target enzyme' results in depletion of essential end-products of the pathway and accumulation of intermediates prior to the blockade. Metabolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay. Such resistance has recently been demonstrated in vitro for the interaction of the tight-binding inhibitor N-phosphonacetyl-L-aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J. Biol. Chem. 255, 11381-11395]. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti-cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5'-monophosphates of 6-azauridine, pyrazofurin and 1-(beta-D-ribofuranosyl)-barbituric acid] with OMP decarboxylase; 5-fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.

Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase.

In Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation, the bulk of the carbamoyl phosphate of the cell is consumed for the biosynthesis of pyrimidines. As a consequence, there is little substrate available for arginine synthesis and the cell growth is impeded. Suppression of arginine auxotrophy by mutations which block aspartate transcarbamoylase activity provides a positive selection technique for mutant strains defective in this enzyme activity. A genetic analysis was performed on 29 mutant strains harboring defects in the structural gene pyrB, encoding the catalytic chains of aspartate transcarbamoylase of Escherichia coli. Extracts from 15 strains contained intact, inactive enzyme-like molecules of the same size as the purified wild type enzyme. These same extracts contained a predominant polypeptide chain which migrated electrophoretically at the same rate as catalytic chains from wild type enzyme. In addition to these 15 different missense mutants, 14 others (presumably chain-terminating mutants) were isolated; no polypeptides corresponding to full length catalytic chains were detected in these strains. Based on their reversion and suppression properties, seven were designated as frameshift and two as amber nonsense. A fine structure recombination map of the pyrB locus was constructed from a series of three-factor transductional crosses. Mutational sites were correlated with regions in the polypeptide sequence by relating their map positions to that of mutation pyrB231 which results in an amino acid replacement at position 128. Moreover, since recent crystallographic studies indicate that residue 128 is located near the junction between the NH2- and COOH-terminal folding domains, the mutational sites can be placed within either of these two regions of tertiary structure. Interallelic complementation experiments showed four units of complementation. Those defining the alpha and beta units were missense mutants with their mutational sites in the NH2- and COOH-terminal domains, respectively. The mutants determining the delta and gamma units involved premature polypeptide chain termination and their mutational sites were correlated with distal regions of the two respective domains. Several mutants of the chain-terminating type failed to complement members of more than one unit. Possible effects of the various mutations and their implications for mechanisms of complementation and enzyme activity are presented.

Developmental changes of citrullinogenesis, mitochondrial N-acetylglutamate content and N-acetylglutamate synthetase in fetal and neonatal rats

A low citrullinogenesis (less than 60 per cent of the adult value) was observed throughout the suckling period when mitochondria isolated from newborn rat liver were incubated in vitro with L-glutamate or succinate as oxidizable substrates. The adult value was reached after weaning. From birth to weaning, intact mitochondria synthesized more citrulline when supplemented with L-glutamate than with succinate. The low citrullinogenesis could not be explained by low carbamoylphosphate synthetase-I and ornithine transcarbamoylase activities that reached adult values at birth. The decreased citrullinogenesis seen for the first three days of life seemed to be related to the low intramitochondrial concentration of N-acetylglutamate, an activator of the carbamoylphosphate synthetase-I. The concentration of this activator did not differ from that reported for adult rat liver mitochondria after the fourth day of life. The discrepancy between the normal value of N-acetylglutamate concentration and the low activity of the N-acetylglutamate synthetase (15 to 30 per cent of the adult activity) is discussed on the basis of acetyl-CoA or L-glutamate availability in mitochondria isolated from newborn or young rats.

Pyrimidine biosynthesis in Plasmodium berghei

1. 1. The complete enzyme sequence of de novo pyrimidine biosynthesis has been found in crude extracts of Plasmodium berghei. 2. 2. Carbamoylphosphate synthase (CPS'ase) utilises l-glutamine and not ammonia as the amine group donor. 3. 3. Aspartate transcarbamoylase (ATC'ase) activity was maximal at pH 9.1 and exhibited normal Michaelis-Menten kinetics with both substrates; apparent K m s were 0.47 mM for Carbamoylphosphate and 2.38 mM for l-aspartate. 4. 4. No purine or pyrimidine metabolite was found to affect the activity of ATC'ase. 5. 5. A range of dicarboxylic acids and a proposed analogue of the transition state, N-( Phosphonacetyl)- l-aspartate (PALA) were found to be inhibitory. The inhibition constant for PALA was 0.48 μM. 6. 6. CPS'ase and ATC'ase both elute in the void volume of Agarose A-5 M and Sephadex G-200 gel columns.

Regulation of Pathways of Ornithine Metabolism

There is a significant increase in four of the urea cycle enzymes in liver from hypothyroid rats, arginase alone showed an opposite trend; these changes are reversed by physiological doses of thyroxine; hyperthyroidism results in a significant decrease in ornithine transcarbamoylase activity. The pattern of change in thyroidectomized and alloxan-diabetic rats showed marked similarities in respect to urea cycle and ornithine-metabolizing enzymes which are discussed in the light of the common feature of hypoinsulinism of diabetes and depressed response to insulin in hypothyroidism. The profile of ornithine-metabolizing enzymes is consonant with the decreased protein synthesis and turnover in hypothyroidism.

Multihormonal control of enzyme clusters in rat liver ontogenesis II. Role of glucocorticosteroid and thyroid hormone and of glucagon and insulin

The role of glucocorticosteroid and thyroid hormone and of glucagon and insulin in the pre- and postnatal developmental formation of carbamoyl-phosphate synthase, ornithine transcarbamoylase, arginase, glutamate dehydrogenase, tyrosine aminotransferase, glucose-6-phosphatase, hexokinase and glucokinase activities in rat liver was investigated. Glucocorticosteroids and a low insulin/glucagon ratio always stimulate formation of carbamoyl-phosphate synthase, ornithine transcarbamoylase, arginase, glutamate dehydrogenase, tyrosine aminotransferase and glucose-6-phosphatase, while glucocorticosteroids and a high insulin/glucagon ratio stimulate formation of glucokinase. Thyroid hormone stimulates the formation of carbamoyl-phosphate synthase, arginase and tyrosine aminotransferase only before birth, whereas it stimulates the formation of glutamate dehydrogenase and glucose-6-phosphatase both before and after birth. Ornithine transcarbamoylase activity is depressed after thyroid-hormone treatment before and after birth. DNA content is always decreased by glucocorticosteroids and increased by thyroid hormone. The effect of these hormones on hexokinase is complex, probably due to different responses of the constitutive isozymes. With the exception of the effects of thyroid hormone on carbamoyl-phosphate synthase, arginase and tyrosine aminotransferase before birth, which may be indirect, the responses of enzyme activities and DNA content to treatment with glucocorticosteroid hormones, glucagon, insulin and thyroid hormone are qualitatively the same in fetuses, neonates, sucklings, weanlings and adults. Thus, the developmental profiles of the enzyme clusters reflect the changing levels of the relevant hormones. The enzymes that are stimulated by glucocorticosteroids and the insulin/glucagon ratio show increases in enzyme activity perinatally and around weaning, and relatively low activities in between, while those enzymes that are additionally stimulated by thyroid hormone differ in exhibiting relatively high activities between birth and weaning.

Changes in the activities of the enzymes of urea synthesis caused by dexamethasone and dibutyryladenosine 3′:5′-cyclic monophosphate in foetal rat liver maintained in organ culture

Liver explants from 19-day foetal rats were maintained in organ culture, in a defined medium, for up to 48h. Both 6-N,2'-O-dibutyryl cyclic AMP, in the presence of theophylline, and dexamethasone caused an increase in the activities of carbamoyl phosphate synthase, argininosuccinate synthetase, argininosuccinate lyase and arginase. These increases could be abolished by simultaneously incubating the explants with cycloheximide. No change in the activity of ornithine transcarbamoylase was found with either hormone. Previous work has shown that injection of corticosteroids into 19.5-day foetal rats in utero did not cause an increase in the arginine synthetase system. Present results suggest that this lack of effect is not due to any incompetence of the foetal rat liver at this stage to respond to this agent. The observations on ornithine transcarbamoylase activity suggest that this enzyme is induced in the liver of the perinatal rat by neither corticosteroids nor hormones acting via cyclic AMP, and it may be that all the enzymes of the urea cycle are induced physiologically by an agent or agents as yet unidentified.

The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.

Reversible dissociation of a carbamoyl phosphate synthase-aspartate transcarbamoylase-dihydroorotase complex from ovarian eggs of Rana catesbeiana: effect of uridine triphosphate and other modifiers.

Glutamine-dependent carbanoyl phosphate synthase [ATP6carbamate phosphotransgerase (dephosphorylating), EC 2.7.2.9], aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) and dihydroorotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3), are copurified as a high-molicular-weight complex from extracts of unfertilized eggs of Rana catesbeiana. UTP is required to maintain the integrity of the complex during the last two purification steps. Removal of the nucleotide results in dissociation of the complex. Based on sedimentation behavior in glycerol gradients, the dissociated carbamoyl phosphate synthase has an apparent molecular weight of 260,000 +/- 20,000 and that of dihydroorotase is estimated at 280,000 +/- 20,000. Aspartate transcarbamoylase is broadly distributed over the gradient. The addition of ATP, 5-phosphoribosyl-1-pyrophosphate, Mg++, or inorganic phosphate to the dossociated complex results in the appearance of a peak of aspartate transcarbamoylase activity with an apparent molecular weight of 110,000 +/- 10,000. Icubation of a mixture of the dissociated enzymes with UTP and Mg++ leads to their reassociation into the high-molecular-weight complex.

Serum Enzymes and Isozymes

The diagnostic use of serum enzyme determinations was evaluated in 33 patients with pulmonary embolisms. The level of serum glutamic oxaloacetic transaminase (SGOT) was elevated in one third and lactate dehydrogenase (LDH) activity was elevated in two thirds of the patients. Elevations of serum LDH activity depended substantially on increases in either LDH 1 or LDH 5 isozymes. In conjunction with increases of serum ornithine transcarbamoylase and α-hydroxybutyrate dehydrogenase activity, these findings indicate extrapulmonary enzyme release into the serum. Most probable enzyme sources are the liver and erythrocytes. Serum creatine kinase level was elevated in five of 22 patients studied, its elevation depending totally on the muscle type (MM) isozyme. The BB isozyme of lung tissue was not found in any of these patients. These results indicate that serum enzyme determinations are of little value in diagnosing pulmonary embolism.

Pyrimidine nucleotide biosynthesis in Phaseolus aureus. Enzymic aspects of the control of carbamoyl phosphate synthesis and utilization.

1. Carbamoyl phosphate synthetase activity of Phaseolus aureus extracts was assayed by coupling it to the catalytic subunit of Escherichia coli aspartate transcarbamoylase and determining the [(14)C]carbamoylaspartate so formed. The stability of the activity was improved by the addition of ornithine and dimethyl sulphoxide to the extraction medium. 2. The synthetase activity was found to utilize either glutamine or ammonia as amino donor, the Michaelis constants being 0.17+/-0.03mm and 6.1+/-1.0mm respectively. N-Acetylglutamate did not significantly alter the rate with either substrate, and azaserine inhibited the reaction with both amino donors to the same extent. 3. Ornithine was shown to stimulate the activity, and to counteract inhibition by UMP. The purine nucleotides IMP and GMP enhanced carbamoyl phosphate formation, whereas AMP had an inhibitory effect. 4. The Michaelis constant for carbamoyl phosphate was determined in concentrated extracts for both aspartate transcarbamoylase and ornithine transcarbamoylase activities, and was 0.13+/-0.03mm and 1.58+/-0.16mm respectively. The ratio of the activities of these two enzymes, determined at near-saturating substrate concentrations, was 1:3 (aspartate transcarbamoylase/ornithine transcarbamoylase). 5. It is concluded that in this plant tissue there is one enzyme, carbamoyl phosphate synthetase, supplying carbamoyl phosphate to both the pyrimidine and arginine pathways, that the pyrimidine pathway claims most of the available carbamoyl phosphate (depending on the concentration of the nucleotide effectors) when this intermediate is present at low concentrations; and that when the carbamoyl phosphate concentration is increased, possibly by ornithine stimulation, a larger proportion can be taken up by the arginine pathway.

Fermentation of agmatine in Streptococcus faecalis: occurrence of putrescine transcarbamoylase.

Putrescine transcarbamoylase, EC 2.1.3.x (carbamoylphosphate:putrescine transcarbamoylase), has been purified from Streptococcus faecalis 10C1 grown on agmatine as primary energy source. The formation of N-carbamoylputrescine from putrescine and carbamoylphosphate serves as a convenient and sensitive assay for this enzymatic activity. The enzyme catalyzes both the phosphorolysis arsenolysis of N-carbamoylputrescine. Arginine does not induce the synthesis of putrescine transcarbamoylase in S. faecalis. Furthermore, the putrescine transcarbamoylase activity is easily separated from ornithine transcarbamoylase activity by gel filtration on Sephadex G-100 indicating that the two activities are associated with different proteins. The significance of this new enzyme in the fermentation of agmatine and its relation to the other known transcarbamoylases are discussed.

Regulation of arginine biosynthesis in Chlamydomonas reinhardii: studies in vivo and of ornithine transcarbamoylase and argininosuccinate lyase activities.

1. Incorporation of [14C]- carbon dioxide into the arginine residue of the protein fraction was used as a measurement of the rate of arginine biosynthesis by the intact cells of Chlamydomonas reinhardii. The rate of synthesis in the presence of exogenous arginine was less than in its absence; the rate by cells grown in the presence of the amino acid was less than by cells grown in its absence. 2. Activities of ornithine transcarbamoylase and argininosuccinate lyase were the same in extracts from cells grown in the presence of arginine as in those from cells grown in its absence. 3. The activity of ornithine transcarbamoylase decreased when the alga was grown more slowly at lower light intensities. 4. The activities of both ornithine transcarbamoylase and argininosuccinate lyase increased when the wild-type alga was deprived of a nitrogen source and when the arginine auxotroph arg-2 was deprived of arginine.

Activation of aspartate transcarbamoylase by purine nucleotides

1. 1. A previous report demonstrated an increase in aspartate transcarbamoylase activity (carbamoylphosphate: l-aspartate carbamoyltransferase, EC 2.1.3.2) upon incubation of an homogenate of Escherichia coli with ATP, GTP, phosphoenolpyruvate, amino acids and magnesium acetate. The object of this study was to ascertain whether enzyme synthesis de novo or enzyme activation was responsible for the increase in aspartate transcarbamoylase activity. 2. 2. Utilizing density labeling and subsequent equilibrium density centrifugation it was demonstrated that the increase in aspartate transcarbamoylase activity was not due to synthesis de novo of this enzyme or to extensive completion of preexisting peptides. 3. 3. It was shown that incubation of an Escherichia coli homogenate with ATP, GTP, phosphoenolpyruvate, amino acids and Mg 2+ resulted in a decrease in the apparent K m of aspartate transcarbamoylase for aspartate while the υ max changed little. 4. 4. Crystalline aspartate transcarbamoylase behaved differently than the enzyme in crude homogenates. Incubation of the crystalline enzyme with ATP, GTP and Mg 2+ caused a threefold increase in υ max . This is the first report of an allosteric effector altering the υ max of aspartate transcarbamoylase. 5. 5. ATP, GTP and Mg 2+ act in a synergistic manner and must be present together for maximum activation of aspartate transcarbamoylase. Previously published studies showed that ATP is an activator and GTP is an inhibitor of this enzyme. 6. 6. Mg 2+ modify the effect of purine nucleotides on aspartate transcarbamoylase activity. GTP in equimolar concentration with Mg 2+ had little effect on enzyme activity, while ATP and equimolar Mg 2+ together were much better activators than ATP alone. 7. 7. The activation of aspartate transcarbamoylase by ATP and GTP in the presence of Mg 2+ reported here may be of biological significance in regulating the relative pool size of pyrimidine nucleotides and purine nucleotides of Escherichia coli.

The inhibition of aspartate transcarbamoylase activity by ATP in a mutant of baker's yeast S1237

The activity of aspartate transcarbamoylase (carbamoyphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) was studies in a partially purified enzyme prepartion from a mutant of baker's yeast S1237. ATP, at the concentration of 2·10 −2 M, was found to inhibit 30% of the aspartate transcarbamoylase activity. Addition of equimolar bicarbonate and glutamine removed this inhibition completely. Kinetics of the mutant enzyme was the same as that of the wild type. The UTP-regulatory site of the mutant enzyme was much more labile than that of the wild type on heating at 50°. These findings suggested that the UTP-regulatory site of the mutant aspartate transcarbamoylase was deformed and also further substantiated the model of Lacroute 3 that aspartate transcarbamoylase and carbamoylphosphate synthase are both present in the same enzyme molecule.