Studies on the availability of intramitochondrial carbamoylphosphate for utilization in extramitochondrial reactions in rat liver

The following observations with isolated mitochondria prepared from rat liver demonstrate that Carbamoylphosphate can readily traverse the mitochondrial membrane: (a) Citrulline synthesis occurs within isolated intact mitochondria at the expense of exogenously added ornithine and [¹⁴C]carbamoylphosphate, providing evidence that the initochondrial membrane does not exclude extramitochondrial car bamoylphosphate from penetrating the intramitochondrial matrix, (b) The [¹⁴C]carbamoylphosphate synthesized within isolated intact mitochondria from NaH¹⁴CO₃ by the action of the N-acetyl-l-glutamate-activated carbamoylphosphate synthetase (CPS-I) is equally available for consumption in intramitochondrial and extramitochondrial reactions, as judged by the coupled activity of CPS-I with either intramitochondrial ornithine carbamoyltransferase or extramitochondrial aspartate carbamoyltransferase. The possibility that the coupled action of CPS-I with intramitochondrial ornithine carbamoyltransferase might prevent the export of carbamoylphosphate into the extramitochondrial medium was also examined. The addition of ornithine to the reaction mixture, at concentrations which are optimal for citrulline production, did not reduce carbamoylphosphate export below$\text{1}\text{3}$ of the total amount of carbamoylphosphate synthesized. These results indicate that the carbamoylphosphate generated intramitochondrially is not compartment ally excluded from participating in cytoplasmic reactions, and raise the possibility that the intramitochondrial carbamoylphosphate synthetase, CPS-I, may be a significant source of the carbamoylphosphate incorporated into hepatic pyrimidines by the cytoplasmic enzymes of the orotate pathway.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.     

Enzymes of the Ornithine-Arginine Metabolism of Trypanosomatids of the Genus Crithidia*

SYNOPSIS. Twelve strains of Crithidia, which fall into 8 species, were tested for occurrence of enzymes of ornithine-arginine metabolism. The following enzymes were investigated: arginase, ornithine carbamoyltransferase, argininosuccinate lyase, citrulline hydrolase, arginine deiminase and urease. Arginase and argininosuccinate lyase were found in all species. Citrulline hydrolase was also found in all but the 2 strains carrying endosymbiotes C. deanei and C. oncopelti. On the other hand, ornithine carbamoyltransferase was found only in these 2 strains. Arginine deiminase and urease were absent in all strains. The existence of a common enzymatic pattern for species of the genus Crithidia is thus reported.     

Arginine metabolism in wineLactobacillus plantarum: in vitro activities of the enzymes arginine deiminase (ADI) and ornithine transcarbamilase (OTCase)

This work was carried out to determine the activity of enzymes involved in arginine metabolism inLactobacillus plantarum isolated from wine and previously characterised at molecular level. The activity of the enzymes arginine deiminase and ornithine transcarbamylase was determined and citrulline and ornithine formed were analysed by HPLC analysis. Although the enzymatic activity was detected in all the strains analysed, a strong variability was observed between strains.Lactobacillus plantrum strain Lp60 is the strain with more possibilities to accumulate citrulline, precursor of the carcinogenic ethyl-carbamate, as showed by its high arginine deiminase activity and low ornithine transcarbamylase activity.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Cyanate specifically inhibits arginine biosynthesis in Escherichia coli K12: a case of by-product inhibition?

Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mM-cyanate. This inhibition could be specifically reversed by arginine. Citrulline (but not ornithine, N-alpha-acetylornithine or N-acetylglutamate) could also restore a normal growth rate. Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine. The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase. A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion. Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

The duplication of arginine catabolism and the meaning of the two ornithine carbamoyltransferases in Bacillus licheniformis.

Arginine deiminase and carbamate kinase activities are shown to be present in cell-free extracts of $\text{Bacillus licheniformis}$. This gives the rationale for the occurrence of an arginine-inducible ornithine carbamoyltransferase in this organism and suggests that, $\text{in vivo}$ and under the proper conditions, this enzyme is able to catalyze the phosphorolysis of citrulline. This also shows that this Bacillus species has two arginine catabolic pathways. This duplication appears to be under the control of O₂.     

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Abstract Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]_0.5) and high cooperativity toward this substrate. We have mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa . Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphisphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.     

Structural and regulatory mutations allowing utilization of citrulline or carbamoylaspartate as a source of carbamoylphosphate in Escherichia coli K-12.

Escherichia coli mutants lacking carbamoylphosphate synthase require arginine and uracil for growth. It is, however, possible to obtain mutants in which carbamoylphosphate is obtained by phosphorolysis of citrulline or carbamyolaspartate. Citrulline utilizers are argG bradytrophs or strains in which the synthesis of ornithine carbamoyltransferase (either of the F or I type) is specifically depressed by unstable chromosomal rearrangements or stable mutations that presumably affect the operators of those genes. Carbamoylaspartate utilization as a source of carbamoylphosphate appears to require more than one mutation; the best-understood strains are pyrD pyrH or pyrC pyrH mutants in which aspartate carbamoyltransferase activity is high and the pool of cytidine triphosphate (feedback inhibitor of aspartate carbamoyl-transferase) is presumably low and in which channeling of carbamoylaspartate towards pyrimidine biosynthesis is considerably reduced. Selection of enzyme overproducers based on a metabolic dependency for a reversed enzymatic reaction can be regarded as a means for isolating regulatory mutants.     

Isolation,identification and genetic organization of the ADI operon in <Emphasis Type="Italic">Enterococcus faecium</Emphasis> GR7

L-Arginine is an indispensable amino acid, as it is required for normal growth of microbes, plants and animals (Szende et al., Cancer Cell Int 1:1475–1480, 2001). Arginine deiminase is the first enzyme of arginine deiminase (ADI) pathway, which catalyzes the conversion of arginine to citrulline and ammonia in an irreversible reaction. Lactic acid bacteria isolated from dairy products were investigated for their ability to hydrolyze arginine. Citrulline production in many LAB strains suggests that the arginine metabolism takes place via the arginine deiminase pathway. The highest arginine deiminase specific activity (0.27 IU/mg) was reported in isolate GR7, which was characterized morphologically, biochemically and by 16S rRNA gene sequencing as Enterococcus faecium. Genetic organization of the ADI operon in E. faecium GR7 was further studied using various molecular biology and computational techniques. Sequence analysis revealed that the genes involved in arginine catabolism are clustered together in an operon (3,906 bp) consisting of the genes arcA (arginine deiminase), arcB (ornithine transcarbamylase), and arcC (carbamate kinase), which are localized on the anti-sense strand of genomic DNA. Nucleotide sequence analysis revealed three open reading frames (ORFs) that were arranged contiguously and transcribed in the same direction, as an apparent operon. The genes followed the order arcC, arcB, arcA, which differs from that found in other microorganisms. The information obtained in this study provides the basis for testing the potential of arginine catabolism to control the emergence of arginine auxotrophic tumors.     

Anabolic ornithine carbamoyltransferase of Escherichia coli and catabolic ornithine carbamoyltransferase of Pseudomonas putida. Steady-state kinetic analysis.

The anabolic and catabolic ornithine carbamoyltransferases of Pseudomonas putida display an undirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro has been previously explained by its kinetic properties, whereas the irreversibility of the catabolic transferase in vivo was shown to be due to its allosteric behaviour. In this work a steady-state kinetic analysis has been carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of Escherichia coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermo-inactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.     

Partial Purification and Properties of Ornithine Transcarbamoylase from Nostoc muscorum Kützing

Ornithine transcarbamoylase (carbamoyl phosphate:l-ornithine carbamoyltransferase, EC 2.1.3.3) has been partially purified from the blue-green alga Nostoc muscorum Kützing, an organism in which the enzyme seems to be involved in a bicarbonate-fixing pathway leading to citrulline. Pertinent to possible regulation of this pathway, the enzyme shows hyperbolic substrate kinetics, has a molecular weight estimated at 75,000 daltons, and its catalytic capability is little influenced by a selection of metabolites that might conceivably act as regulators in vivo. Thus it seems unlikely that this enzyme is the control point for bicarbonate fixation. In terms of energy of activation (12.3 kcal/mole), size and Km for carbamoylphosphate, the Nostoc enzyme resembled preparations from liver and higher plants more than preparations from Streptococcus and Mycoplasma. The enzymes from Streptococcus and Mycoplasma are probably specialized for citrulline breakdown rather than citrulline synthesis. The Km for ornithine was 2.5 mm at a saturating concentration of carbamoylphosphate and the Km for carbamoylphosphate was 0.7 mm at an ornithine concentration of 2 mm. Ornithine was inhibitory at concentrations greater than 2 mm. Phosphate was a competitive inhibitor with respect to carbamoylphosphate. The pH optimum for citrulline synthesis was 9.5.     

Arginine catabolism in Lactobacillus sake isolated from meat

Lactobacillus sake isolated from meat can hydrolyze arginine via the arginine deiminase pathway. Two enzymes, arginine deiminase and ornithine transcarbamylase, have been revealed by detection of their reaction products, citrulline and ornithine, respectively. The production of citrulline depends on the concentration of glucose in a synthetic medium; it does not occur when the concentration of glucose is 27.5 mM or higher.     

Arginine catabolism in Lactobacillus sake isolated from meat.

Lactobacillus sake isolated from meat can hydrolyze arginine via the arginine deiminase pathway. Two enzymes, arginine deiminase and ornithine transcarbamylase, have been revealed by detection of their reaction products, citrulline and ornithine, respectively. The production of citrulline depends on the concentration of glucose in a synthetic medium; it does not occur when the concentration of glucose is 27.5 mM or higher.     

Enzymes of the Ornithine-Arginine Metabolism of Trypanosomatids of the Genus Herpetomonas*

SYNOPSIS. Five strains of trypanosomatids, Herpetomonas megaseliae, H. samuelpessoai, H. muscarum muscarum, H. muscarum ingenoplastis and a newly isolated Herpetomonas sp., were examined for the enzymes of arginine-ornithine metabolism. Ornithine carbamoyltransferase (E.C. 2.1.3.3) and argininosuccinate lyase (E.C. 4.3.2.1) were detected in cell extracts of H. megaseliae, H. samuelpessoai and H. muscarum muscarum but not of others. Both enzymes seemed repressible by arginine, which could account for their apparent absence in H. muscarum ingenoplastis and Herpetomonas sp., which grow in a complex, arginine-rich medium. Additionally, arginine deiminase (E.C. 3.5.3.6) and citrulline hydrolase were detected in cell extracts of the 5 strains examined. This latter enzyme, previously described only in Tetrahymena, effects the single-step hydrolysis of citrulline into ammonia, ornithine and CO₂. Arginase (E.C. 3.5.3.1) and urease (E.C. 3.5.1.5) were not found in any of the strains examined. Some of the physicochemical characteristics of the enzymes encountered are described.     

Mode of Action of the Toxin from Pseudomonas phaseolicola: II. Mechanism of Inhibition of Bean Ornithine Carbamoyltransferase

A chlorosis-inducing toxin of Pseudomonas phaseolicola was examined for inhibition of ornithine carbamoyltransferease prepared from acetone powder of bean (Phaseolus vulgaris L.) plants. The enzyme has a pH optimum at 8.5, involves a ternary complex reaction mechanism, and shows Michaelis constants of 5.0 mm and 1.7 mm for ornithine and carbamoylphosphate, respectively. Assuming reversible catalysis, Michaelas constants of 11 mm and 3.3 mm are calculated for citrulline and arsenate. Toxin induces allosteric competitive inhibition in relation to carbamoylphosphate and a noncompetitive mode of inhibition in relation to ornithine, except at high toxin concentrations where uncompetitive inhibition is observed. In the backward assay, competitive inhibition is observed for both arsenate and citrulline. Inhibition is increased with preincubation time and shows saturation kinetics with regard to toxin concentration.     

Control of enzyme synthesis in the arginine deiminase pathway of Streptococcus faecalis

The formation of the arginine deiminase pathway enzymes in Streptococcus faecalis ATCC 11700 was investigated. The addition of arginine to growing cells resulted in the coinduction of arginine diminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.3). Growth on glucose-arginine or on glucose-fumarate-arginine produced a decrease in the specific activity of the arginine fermentation system. Aeration had a weak repressing effect on the arginine deiminase pathway enzymes in cells growing on arginine as the only added substrate. By contrast, depending on the growth phase, a marked repression of the pathway by oxygen was observed in cells growing on glucose-arginine. We hypothesize that, in S. faecalis, the ATP pool is an important signal in the regulation of the arginine deiminase pathway. Mutants unable to utilize arginine as an energy source, isolated from the wild type, exhibited four distinct phenotypes. In group I the three enzymes of the arginine deiminase pathway were present and probably affected in the arginine uptake system. Group II mutants had no detectable arginine deiminase, whereas group III mutants had low levels of ornithine carbamoyltransferase. Group IV mutants were defective for all three enzymes of the pathway.     

Intracellular localization of the arginine deiminase pathway inStreptococcus mitis

A subcellular fractionation study was carried out onStreptococcus mitis ATCC 9811 to determine the location of the arginine deiminase pathway. Arginine deiminase activity was detected in the cell wall fraction, ornithine carbamoyltransferase activity was recovered in both the cell wall and cytoplasmic fractions, and carbamate kinase activity was detected in the cytoplasmic fraction.