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Saccharomyces cerevisiae mutants lacking the anabolic L-threonine deaminase, the ilvl mutants, have been found to exhibit a normal ability to grow, without auxotrophy towards isoleucine, on L-threonine or L-serine as only nitrogen nutrient. Starting from a strain carrying a ilvl mutation, a new mutation affecting the ability to utilize L-threonine as nitrogen source was selected. This mutation, which also impairs the ability to utilize L-serine, has been denominated cha, for 'catabolism of hydroxyamino acids' and was found to result in the lack of a catabolic L-serine (L-threonine) deaminase. This enzyme which, unlike the anabolic threonine deaminase, is more active towards serine than towards threonine, differs from the latter enzyme by a number of biochemical and regulatory properties. Whereas the anabolic enzyme is an allosteric enzyme sensitive to feedback inhibition by isoleucine, the catabolic enzyme exhibits Michaelian kinetics: no control of its activity has been detected. Its synthesis is induced by L-serine and L-threonine. These two enzymes, which thus can be easily differentiated by means of their regulations, display a limited ability to compensate for one another's absence and appear to play clearly distinct roles under normal physiological conditions. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae L-asparagine auxotrophy, resulting from a lack of L-asparagine synthesis, needs simultaneous defects in two genes. This unusual situation is shown to result from the occurrence of two L-asparagine synthetases. The meaning of this duplication remains obscure. The properties of the two enzymes are remarkably similar: both have a molecular weight of 150 000 and they exhibit a slight repression and a similar inhibition by asparagine. Neither one is located in mitochondria and both are probably cytosolic. Their identification so far lies only in their behaviour on ion-exchange chromatography and in the encoding genes. [ABSTRACT FROM AUTHOR]

L-Asparagine synthesis in Saccbaromyces cerevisiae is performed by a glutamine-dependent asparagine synthetase of the type found in higher organisms. Auxotrophy for asparagine has been obtained in two classes of mutants. In class I, asparagine synthetase activity is cancelled. These mutants combine two mutations, asnA- and asnB-. Neither asnA- nor asnB- mutation alone leads to total auxotrophy. Partial auxotrophy as well as a strong decrease in enzyme activity result from asnA- mutation. No change is detectable in cells with the asnB- mutation alone. This, and Jones' report [J. Bacteriol. 134, 200-207 (1978)] of auxotrophy resulting from the combination of two mutations, are strong supports for asparagine synthesis being an unusual biosynthetic operation. In class II, auxotrophy results from a single mutation which leads to a modification of the efficiency of the asparaginyl-tRNA synthetase (asnRS- mutation). This auxotrophy is cancelled if asparaginase I activity (the only one present in Ε 1278b wild type) is cancelled by casnl- mutation. This latter mutation allows an increase in the asparagine pool which is able to compensate for the asparaginyl-tRNA synthetase partial defect of the asnRS- mutant. [ABSTRACT FROM AUTHOR]

Ornithine, citrulline and homoarginine having a regulatory role on arginine biosynthetic enzymes in wild-type, but not in argR mutants, do not interfere with aminoacylation of tRNAArg in vitro or in vivo. The same aminoacylation reaction does not seem to be modified in argR mutations, strengthening the hypothesis of a mutation, involving the apopressor. [ABSTRACT FROM AUTHOR]

The regulation of arginine biosynthetic enzymes in yeast is subjected to a double control. One level of arginine enzyme synthesis is under the control of an apo-repressor, called ARGR. ARGR molecules control specifically the arginine pathway. A second level of control of arginine biosynthesis has been disclosed. It also controls tryptophan, histidine, lysine, isoleucine-valine and leucine, and probably many more biosynthesis. The general mechanism is turned on a leaky mutants in any of the amino acid pathways mentioned above. [ABSTRACT FROM AUTHOR]

L-Ornithine carbamoyltransferase and arginase from Saccharomyces cerevisiae have been extensively purified and their quaternary structures have been determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Subunit cross-linking with glutaraldehyde shows that both enzymes have trimeric structures. Ornithine carbamoyltransferase is built up by the association of three subunits of molecular weight 37500; arginase is made of three subunits of 39000. The latter observation has lead us to reinvestigate the quaternary structure of rat liver arginase for which, in spite of an earlier report to the contrary [H. Hirsch-Kolb and D. Greenberg, J. Biol. Chem. 243, (1968) 6123-6129], evidence of a trimeric structure is also obtained. The data suggest that the 1-to-1 regulatory complex of ornithine carbamoyltransferase and arginase of S. cerevisiae, leading to ornithine carbamoyltransferase inhibition, is an hexamer. [ABSTRACT FROM AUTHOR]

Arginase synthesis appears to be under the control of at least two distinct processes. In addition to substrate induction which operates through a Jacob and Monod mechanism, one may define a process of nitrogen catabolite repression which is a part of the ammonium effect. The two processes show a large degree of independence most specifically shown by mutations which independently cancel each of the two mechanisms without affecting the gross physiology. In this line we show that controversial conclusions cm be reached when using different approaches to provoke catabolite derepression. The nitrogen catabolite repression is abolished by the gdhA- mutation which affects the structural gene for the NADP-specific glutamate dehydrogenase. The effect of the gdhA- mutation is retained when the metabolic defect is compensated by addition of glutamate as well as the best nitrogen nutrients. glutamine and asparagine. This control of arginase and of some, but not all, other nitrogen catabolic enzymes may operate at constant levels of glutamate dehydrogenase (NADP). It occurs when the glutamate dehydrogenase (NADP) is in its catalytically active state, which requires the simultaneous presence of two substrates. NH4+ and 2-oxoglutarate. In fungi the glutamate dehydrogenase (NADP) is the first enzyme of the assimilatory pathway of ammonium. which, when functioning, makes arginase unnecessary. However the regulatory function seems to operate by an intrinsic modification of the enzyme itself rather than by the result of its catalytic action. Carbon catabolite repression appears largely independent from nitrogen catabolite repression and vice versa. However. arginase synthesis is enhanced in mutants lacking aconitase as well as by very poor carbon nutrition. Both conditions decrease the level of 2-oxoglutarate which is required for the expression of nitrogen catabolite repression. Once specific induction and nitrogen catabolite repression are clearly defined. it becomes possible to show that additional regulatory processes are involved in arginase synthesis (Wiame, 1973). [ABSTRACT FROM AUTHOR]

The cargB or CAR2 gene, coding for ornithine aminotransferase, was isolated by functional complementation of a cargB- mutation in Saccharomyces cerevisiae. It was used as a hybridization probe to analyse RNA and chromosomal DNA from four strains bearing cis-dominant regulatory mutations leading to constitutive, matingtype-dependent, ornithine aminotransferase synthesis. The four mutations appear to be insertions. Their size and restriction pattern suggested that they were transposable elements, Ty1. All were inserted in the same orientation with respect to the cargB gene. We cloned the cargB gene with its associated insertion from two constitutive mutants (cargB+Oh-I and cargB+Oh-2). We determined the sequence of the cargB 5' region from the wild-type gene and from the two mutated genes. The DNA sequences of the extremities of the two insertions were very homologous but not identical and were similar to previously reported Ty1 element direct repeats (δ). The same five-base-pair sequence, ATATA, was found at both ends of both Ty1 elements, indicating that both Ty1 were transposed to the same site. This site is located 115 base pairs upstream from the putative cargB coding region. The 5' end of cargB transcript as determined by SI mapping was the same in the wild-type strain and in the four mutants. The cargB transcript was not detected in the wild-type strain grown under non-induced conditions, while under the same conditions it was present in all four mutants. [ABSTRACT FROM AUTHOR]

Points out that in Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. Observation that these microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity; Indication that the competitive pattern of this inhibition leads to its being inoperative in ornithine-grown cells, where the intracellular concentration of ornithine is high.

We present genetic evidence for the enzymes 4-aminobutyrate:2-oxoglutarate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase [NAD(P)+] (EC 1.2.1.16) constituting the functional pathway for the utilization of 4-aminobutyric acid as a nitrogen source by Saccharomyces cerevisiae. We show that the pathway is induced by 4-aminobutyric acid and that the presence of the pathway enzymes probably requires the integrity of a positive control element. [ABSTRACT FROM AUTHOR]

Glutamine synthetase activity is modulated by nitrogen repression and by two distinct inactivation processes. Addition of glutamine to exponentially grown yeast leads to enzyme inactivation, 50% of glutamine synthetase activity is lost after 30 min (a quarter of the generation time). Removing glutamine from the growth medium results in a rapid recovery of enzyme activity. A regulator mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism. The gdhCR mutation is also increases the level of proteinase B in exponentially grown yeast. Inactivation of glutamine synthetase is also observed during nitrogen starvation. This inactivation is irreversible and consists very probably of a proteolytic degradation. Indeed, strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation. [ABSTRACT FROM AUTHOR]

A careful enzyme specificity analysis has revealed the presence of a typical γ-glutamyltranspeptidase in the yeast Saccharomyces cerevisiae. The enzyme cellular level is low in the presence of NH4+ as a sole nitrogen source and rises when individual amino acids are used as nitrogen sources. The γ-glutamyltranspeptidase appears to be repressed by NH4+ and escapes to the regulatory circuits under the control of glutamine and the glutamate-dehydrogenase · NH4+ complex. The transpeptidase cellular level is unaffected in mutants which have lost the general amino acid permease and specific systems for L-arginine and L-lysine. In contrast, a low enzyme level is observed when growing an apf mutant on urea; this mutant is most probably affected in a common element shared by all the amino acid permeation systems. Urea appears to be a nitrogen source which promotes a high transpeptidase level in the wild-type strain. The reported data are discussed in the light of the current theories about the intervention of glutathione metabolism in the translocation of amino acids. [ABSTRACT FROM AUTHOR]

The reaction catalyzed by ornithine carbamoyltransferase can participate in either the anabolism or the catabolism of arginine. The carbamoylation of ornithine, yielding citrulline, is involved in the biosynthetic sequence; the reverse reaction, the phosphorolysis of citrulline, is the second step of the arginine deiminase pathway. The ornithine carbamoyltransferases of a number of microorganisms which can fulfil both of these functions have been studied in this work. This group of organisms was found to possess two distinct ornithine carbamoyltransferases. The functions of these enzymes were surmised by determining the type of genetic regulation to which they were subjected. The kinetic properties of these various enzymes have been determined. All of them, regardless of the role they play in the cell, catalyze both the synthesis and arsenolysis of citrulline. The anabolic transferase of Pseudomonas is the only enzyme which displays functional irreversibility. A comparison of the quaternary structure of these transferases was performed and reveals interesting features in relation to the metabolic function of these enzymes. All well-characterized anabolic enzymes have low molecular weights (from 150 000-105 000) and are likely to be trimers. Catabolic enzymes, with the exception of those of Bacillus licheniformis and Halobacterium salinarium, display much higher molecular weights and more elaborate quaternary structure. The properties of these two groups of transferases are discussed in relation to their metabolic role in the cells. [ABSTRACT FROM AUTHOR]

Partial fractionation of Saccharomyces cerevisiae spheroplast lysate shows that arginase, ornithine carbamoyltransferase and carbamoylphosphate synthetase have the same distribution as the cytosol marker enzyme glucose-6-phosphate dehydrogenase. The same holds true in stepwise homogenization experiments of spheroplast lysates of S. cerevisiae. This second method applied to Debaryomyces hansenii, Hansenula anomala and Schizosaccharomyces pombe shows, in contrast, that ornithine carbamoyltransferase is particulate in these yeasts. Its solubilization is in line with that of citrate synthase taken as mitochondrial matrix marker, whereas arginase acts like glucose-6-phosphate dehydrogenase. In Schizosaccharomyees pombe carbamoylphosphate synthetase is particulate too. Density gradient centrifugation of a particulate fraction of Schizosaceharomyces pombe suggests that ornithine carbamoyltransferase and carbamoylphosphate synthetase are associated with the mitochondria. In Saccharomyces cerevisiae and in some other yeasts [Messenguy & Wiame (1969) FEBS Lett. 3, 47-49; Wiame (1974) Proc. 9th Meet. Fed. Eur. Bioehem. Sot., pp. 161-177] arginase binds to ornithine carbamoyltransferase in the presence of arginine and ornithine, thereby inhibiting its activity but leaving arginase activity unmodified. This regulation by reversible stoechiometric interaction between the two enzymes prevents recycling of ornithine to arginine when the latter is used as a source of nitrogen. Results presented here show a correlation between the shift of ornithine carbamoyltransferase and carbamoylphosphate synthetase from mitochondrial to extramitochondrial fraction and the existence of arginase/ornithine carbamoyltransferase regulatory interaction. This regulation and extramitochondrial ornithine carbamoyltransferase are absent from obligate aerobe yeasts and may reflect an adaptation to the... [ABSTRACT FROM AUTHOR]

A simple procedure is reported for the purification of the two malate synthases of Escherichia coli involved in the use of acetate (A) and glycolate (G), respectively, as carbon source. Both isoenzymes have been isolated simultaneously and in equally high amounts from a strain constitutive for the glyoxylate cycle, after growth on glycolate. After separation from each other by column chromatography on DEAE-cellulose, the malate synthases have been purified by heating, ammonium sulfate precipitation and molecular sieve chromatography on a Sephadex G-200 column, in the constant presence of their specific protectors, glycolate for malate synthase A, and oxalate for malate synthase G. The puritied preparations so obtained appeared to be homogeneous on the basis of analytical ultracentrifugation and moving-boundary electrophoresis. The sedimentation coefficient at infinite dilution was 4.6 S for ma]ate synthase A and 5.2 S for malate synthase G; their electrophoretic mobilities at pH 8 were 4.3×10-5 cm2 V-1 s-1 and 4.1×10-5 cm2 V-1 s-l, respectively. The apparent molecular weight of malate synthase A, estimated by Archibald's method, was 54000; that of malate synthase G, estimated by light scattering, was 52000. Threonine and serine have been found, by Sanger's method, to be N-terminal residues of malate synthases A and G, respectively. [ABSTRACT FROM AUTHOR]

The catabolic ornithine carbamoyltransferase of Pseudomonas is a high molecular weight enzyme which, in vivo, catalyzes the phosphorolysis of citrulline. The enzyme exhibits pronounceed allosteric properties which have been studied in great detail for the reverse direction of the reaction, the carbamoylation of ornithine. The interpretation of the kinetic data according to the “concerted transition” model of Monod, Wyman and Changeux led to the suggestion that tile enzyme is an octamer built, up by the aggregation of identical subunits. The present work was aimed at verifying that point and provided the following observations. Sucrose density gradient sedimentation and polyacrylamide gel electrophoresis of the dissociated protein showed that the enzyme is made up of eight identical subunits with respect to both charge and size. The apparent molecular weight of the subunits is 39000. The number of subunits is eight as determined by crosslinking of the enzyme in the presence of glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The enzyme undergoes a self-associating reaction which is under the control of its substrates, products and allosteric effectors. The study of the effect, of those ligands on the kinetic behavior and on the distribution of the aggregation states of ornithine carbamoyltransferase suggests that the active form of this enzyme is an octamer but that several inactive or less active octameric, tetrameric and dimeric stages exist. [ABSTRACT FROM AUTHOR]

The properties of the catabolic ornithine carbamoyltransferase of Pseudomonas fluorescens are compared to those of the anabolic transferase of the same organism. The comparison is also extended to a mutationally modified enzyme which, in contrast to the catabolic enzyme, is able to synthesize citrulline in vivo. All three activities are measured in the direction of citrulline synthesis. The catabolic ornithine carbamoyltransferase and the mutationally modified enzyme have an approximate molecular weight of 400000 while that of the anabolic enzyme is 100000. The catabolic transferase exhibits a number of regulatory properties which are not observed with the anabolic enzyme: 1. Its saturation by carbamyolphosphate is intensively cooperative. Its half saturation concentration is more than one hundred times higher than the Km of the anabolic enzymes; this explains its inability to perform an anabolic function in vivo. 2. Phosphate, a substrate in the physiological direction of the reaction and a low-energy-level indicator, acts as an activator of the enzyme; it abolishes the cooperativity of the carbamoylphosphate and increases the capacity of the enzymes. 3. ATP, a product of the catabolic pathway and a high-energy4evel signal, reduces the capacity of the enzyme. 4. Putrescine, another product of the catabolic pathway, and its analogs, inhibit the enzyme by decreasing its apparent affinity for carbamoylphosphate. The catabolic transferase of the mutant 1RC204M1 shares all those properties with the wild- type enzyme, but, owing to a reduced cooperativity of carbamoylphosphate, it is able to perform an anabolic function at reduced rate. [ABSTRACT FROM AUTHOR]

The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control of arginine and ornithine, involves a regulatory site on the ornithine carbamoyltransferase distinct from its catalytic site. This regulatory site is responsible for the partial inhibition of ornithine carbamoyltransferase by excess of ornithine. The strong inhibition of ornithine carbamoyltransferase by arginase in the presence of arginine requires the integrity of the mechanism which leads to partial inhibition by excess of ornithine. [ABSTRACT FROM AUTHOR]