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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]

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]

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]