Organization and control in the arginine biosynthetic pathway of Neurospora.

Eight enzymes involved in the conversion of acetylglutamate to arginine in Neurospora crassa were studied. The data indicate that of three enzymes early in the sequence, only the first, acetylglutamate kinase, is a nonorganellar enzyme. The next two, N-acetyl-gamma-glutamyl-phosphate reductase and acetylornithine aminotransferase, are in the mitochondrion, which was previously shown to contain the subsequent enzymes: acetylornithine-glutamate acetyltransferase, ornithine carbamyltransferase, and carbamyl-phosphate synthetase A (arginine specific). The last two enzymes of the pathway, argininosuccinate synthetase and argininosuccinate lyase, were previously shown to be cytosolic. All enzymes but one have low amplitudes or repression. Their levels respond little to arginine excess and are about twofold elevated (threefold for ornithine carbamyltransferase) as a result of arginine limitation in the arg-12-8 strain. No restriction of the incorporation of mitochondrial enzymes into mitochondria could be detected when the levels of these enzymes were elevated. Two enzymes, acetylglutamate kinase and carbamyl-phosphate synthetase A, which initiate the synthesis of the ornithine and guanidino moieties of arginine, respectively, show the lowest specific activities in crude extract. These enzymes display special regulatroy features. Acetylglutamate kinase, which has a typically low amplitude of repression, is subject to feedback inhibition. Carbamyl-phosphate synthetase A is wholly insensitive to arginine or citrulline in vitro or in vivo, but displays a very large amplitude of repression (about 60-fold). It is unique in that it can be almost completely repressed by growth of mycelia in excess arginine. These data suggest that mitochondrial localization may be incompatible with a mechanism of feedback inhibition by a cytosolic effector, arginine. Further, they suggest that the high repressibility of carbamyl-phosphate synthetase A compensates for its feedback insensitivity.     

Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes.

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.     

Control of the ornithine cycle in Neurospora crassa by the mitochondrial membrane.

In Neurospora crassa, the mitochondrial membrane separates ornithine used in arginine biosynthesis from ornithine used in the arginine degradative pathway in the cytosol. Ornithine easily exchanges across the mitochondrial membrane under conditions appropriate for synthesis of the immediate biosynthetic product, citrulline. Neither of the two mitochondrial enzymes required for the ornithine-to-citrulline conversion is feedback inhibitable in vitro. Nevertheless, when arginine is added to cells and cytosolic ornithine increases as arginine degradation begins, the rate of citrulline synthesis drops immediately to about 20% of normal (B. J. Bowman and R. H. Davis, Bacteriol. 130:285-291, 1977). We have studied this phenomenon in citrulline-accumulating strains carrying the arg-1 mutation. Citrulline accumulation is blocked when arginine is added to an arg-1 strain but not to an arg-1 strain carrying a mutation conferring insensitivity of intramitochondrial ornithine synthesis to arginine. Thus, ornithine is evidently unable to enter mitochondria in normal (feedback-sensitive) cells. Other experiments show that cytosolic ornithine enters mitochondria readily except when arginine or other basic amino acids are present at high levels in the cells. We conclude that in N. crassa, the mitochondrial membrane has evolved as a secondary site of feedback inhibition in arginine synthesis and that this prevents a wasteful cycling of catabolic ornithine back through the anabolic pathway. This is compared to the quite different mechanism by which the yeast Saccharomyces cerevisiae prevents a futile ornithine cycle.     

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.     

Acetylglutamate kinase: a feedback-sensitive enzyme of arginine biosynthesis in Neurospora

A radioactive assay was developed for the arginine-synthetic enzyme, acetylglutamate kinase (EC 2.7.2.8). Activity of the enzyme was demonstrated in crude extracts of Neurospora mycelium. Precipitation with ammonium sulfate, resulting in separation of the enzyme from an inhibitor, was initially required to detect activity. Most preparations are only partially sensitive to arginine, with maximal inhibition achieved at an effector concentration of 0.5 mM. The enzyme is activated about 10% by 1 mM lysine or citrulline, while 1 mM ornithine stimulates activity by 75%.     

A single precursor protein for two separable mitochondrial enzymes in Neurospora crassa

The arg-6 locus of Neurospora crassa encodes two early enzymes of the arginine biosynthetic pathway, acetylglutamate kinase and acetylglutamyl-phosphate reductase. Previous genetic and biochemical analyses of this locus and its products showed that: 1) strains carrying polar nonsense mutations in the acetylglutamate kinase gene lacked both enzyme activities (Davis, R.H., and Weiss, R.L. (1983) Mol. Gen. Genet. 192, 46-50), and 2) the proteins isolated from mitochondria were completely separable (Wandinger-Ness, A., Wolf, E.C., Weiss, R.L., and Davis, R.H. (1985) J. Biol. Chem. 260,5974-5978). These data suggested that the two enzymes were initially synthesized as a single precursor which was subsequently cleaved into two distinct polypeptides. We report here the identification of a high molecular weight protein, synthesized in vitro from isolated N. crassa RNA, that contains sequences corresponding to acetylglutamate kinase as well as acetylglutamyl-phosphate reductase. An analogous precursor was identified in vivo by pulse-labeling experiments. The precursor was similar to other mitochondrial precursors in that its uptake and processing in vivo was rapid and required an intact mitochondrial electrochemical gradient. This represents the first report of a bifunctional protein precursor which gives rise to two mitochondrial enzymes.     

Acetylglutamate kinase. A mitochondrial feedback-sensitive enzyme of arginine biosynthesis in Neurospora crassa

The radioisotopic method used to assay acetylglutamate kinase (EC 2.7.2.8) of Neurospora crassa has been shown to detect two distinct enzymatically catalyzed reactions. The enzymes were separated by differential centrifugation into a cytosolic activity and an organellar activity. Both activities required ATP and were thermal-labile. The cytosolic activity was insensitive to inhibition by arginine and formed a stable reaction product in the absence of hydroxylamine. The organellar activity had an absolute requirement for hydroxylamine in order to form a stable reaction product. The product of the cytosolic activity was separated from acetylglutamate hydroxamate (the product of the organellar activity) and was identified as the cyclic amide pyroglutamate by cation exchange chromatography. The organellar activity has been implicated in arginine biosynthesis by the following criteria: it was completely and specifically inhibited by arginine concentrations as low as 200 microM; its level was elevated 2-fold in a mutant strain with derepressed levels of arginine biosynthetic enzymes; and it was absent in an arginine auxotrophic strain (the cytosolic activity was present). The organellar activity co-sedimented with mitochondria during isopycnic gradient centrifugation. The metabolic problems posed by a mitochondrial location of a feedback-sensitive enzyme and the cytosolic location of its effector are discussed.     

Mitochondrial steps of arginine biosynthesis are conserved in the hydrogenosomes of the chytridiomycete Neocallimastix frontalis

Arginine biosynthesis in eukaryotes is divided between the mitochondria and the cytosol. The anaerobic chytridiomycete Neocallimastix frontalis contains highly reduced, anaerobic modifications of mitochondria, the hydrogenosomes. Hydrogenosomes also occur in the microaerophilic flagellate Trichomonas vaginalis, which does not produce arginine but uses one of the mitochondrial enzymes, ornithine transcarbamoylase, in a cytosolic arginine dihydrolase pathway for ATP generation. EST sequencing and analysis of the hydrogenosomal proteome of N. frontalis provided evidence for two mitochondrial enzymes of arginine biosynthesis, carbamoylphosphate synthase and ornithine transcarbamoylase, while activities of the arginine dehydrolase pathway enzymes were not detectable in this fungus.     

Ornithine decarboxylase from Neurospora crassa. Purification, characterization, and regulation by inactivation

Ornithine decarboxylase, a highly regulated enzyme of the polyamine pathway, was purified 670-fold from mycelia of Neurospora crassa that were highly augmented for enzyme activity. The enzyme is significantly different from those reported from three other lower eucaryotic organisms: Saccharomyces cerevisiae, Physarum polycephalum, and Tetrahymena pyriformis. Instead, the enzyme closely resembles the enzymes from mammals. The Mr = 110,000 enzyme is a dimer of 53,000 Da subunits, with a specific activity of 2,610 mumol per h per mg of protein. Antisera were raised to the purified enzyme and were rendered highly specific by cross-absorption with extracts of a mutant strain lacking ornithine decarboxylase protein. With the antisera, we show that the inactivation of the enzyme in response to polyamines is proportional to the loss of ornithine decarboxylase protein over almost 2 orders of magnitude. This is similar to the inactivation process in certain mammalian tissues, and different from the process in S. cerevisiae and P. polycephalum, in which enzyme modification, without proportional loss of antigen, accompanies enzyme inactivation. The N. crassa enzyme is therefore suitable as a microbial model for studies of the molecular regulation of the mammalian enzyme.     

The N-acetylglutamate synthase/N-acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesis.

In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869-42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.     

Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis.

Streptococcus lactis metabolizes arginine by the arginine deiminase (ADI) pathway. Resting cells of S. lactis grown in the presence of galactose and arginine maintain a high intracellular ornithine pool in the absence of arginine and other exogenous energy sources. Addition of arginine results in a rapid release of ornithine concomitant with the uptake of arginine. Subsequent arginine metabolism results intracellularly in high citrulline and low ornithine pools. Arginine-ornithine exchange was shown to occur in a 1-to-1 ratio and to be independent of a proton motive force. The driving force for arginine uptake in intact cells is supplied by the ornithine and arginine concentration gradients formed during arginine metabolism. These results confirm studies of arginine and ornithine transport in membrane vesicles of S. lactis (A. J. M. Driessen, B. Poolman, R. Kiewiet, and W. N. Konings, Proc. Natl. Acad. Sci. USA, 84:6093-6097). The activity of the ADI pathway appears to be affected by the internal concentration of (adenine) nucleotides. Conditions which lower ATP consumption (dicyclohexylcarbodiimide, high pH) decrease the ADI pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity. The arginine-ornithine exchange activity matches the ADI pathway most probably by adjusting the intracellular levels of ornithine and arginine. Regulation of the ADI pathway and the arginine-ornithine exchanger at the level of enzyme synthesis is exerted by glucose (repressor, antagonized by cyclic AMP) and arginine (inducer). An arginine/ornithine antiport was also found in Streptococcus faecalis DS5, Streptococcus sanguis 12, and Streptococcus milleri RH1 type 2.     

Isolation and characterization of Neurospora crassa mutants impaired in feedback control of ornithine synthesis.

Thirty-two independent mutants were isolated which overcame the proline requirement of pro-3 mutations in Neurospora crassa. The mutations were not revertants, appeared to be allelic, were closely linked or allelic to arg-6, and in strains unable to degrade ornithine no longer suppressed the proline requirement. The suppressor mutations did not alter the levels of biosynthetic or catabolic enzymes, yet allowed accumulation of ornithine. Suppressed strains unable to degrade arginine still produced ornithine (as detected by growth) in arginine-supplemented medium. The results suggest that the suppressor mutants were impaired in the feedback inhibition of ornithine synthesis by arginine. The activity of the appropriate biosynthetic enzyme was less sensitive to inhibition by arginine. The potential usefulness of such mutations is discussed.     

Ornithine transcarbamylase from Neurospora crassa: purification and properties

Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.     

Simultaneous purification of three mitochondrial enzymes. Acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from Neurospora crassa

The early enzymes of arginine biosynthesis in Neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline. The final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm. We have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract. Acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) were purified to homogeneity and near homogeneity, respectively. The large catalytic subunit of carbamoyl-phosphate synthetase was also purified to homogeneity. The three enzymes were resolved into separate fractions by chromatography on three dye-ligand affinity resins, which are specific for nucleotide binding enzymes and have a high protein binding capacity. High performance liquid chromatography was employed in the final stages of purification and was extremely effective in fractionating both acetylglutamate kinase and acetylglutamyl-phosphate reductase from proteins with very similar properties, which were not removed by other techniques. The purified proteins were used to raise specific antisera against these proteins. Acetylglutamate kinase and acetylglutamyl-phosphate reductase were shown to be immunologically unrelated. This finding suggests that the arg-6 locus encompasses two nonoverlapping cistrons. The antisera raised against carbamoyl-phosphate synthetase has been shown to cross-react with related enzymes in Saccharomyces cerevisiae, Escherichia coli, and rat liver (Ness, S. A., and Weiss, R. L. (1985) J. Biol. Chem. 260, 14355-14362). Acetylglutamate kinase is a regulatory enzyme and has been shown to be feedback-inhibited by arginine. We have determined the submitochondrial localization of acetylglutamate kinase and the second arg-6 product, acetylglutamyl-phosphate reductase. Both enzymes were shown to be soluble matrix enzymes. We discuss the relevance of this finding with respect to possible mechanisms for end product inhibition of a mitochondrial enzyme by a cytoplasmic effector.     

Identification of an arginine carrier in the vacuolar membrane of Neurospora crassa

A number of arginine derivatives were tested for their ability to inhibit arginine uptake into vacuolar membrane vesicles of Neurospora crassa. The guanido side chain and L-configuration were found to be important for recognition by the arginine carrier. Based upon the specificity of recognition, a reactive arginine derivative (N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane) was synthesized which has an intact guanido side chain and a diazo group at the carboxyl end. The latter decomposes to a reactive carbene group. This derivative inhibited arginine uptake into vacuolar membrane vesicles at low concentrations. Radioactive N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane was covalently bound to vacuoles. Binding was specific for a single membrane protein with an approximate molecular weight of 40,000, saturable (2 pmol/mg vacuolar membrane protein), and inhibited by 100 mM L-arginine but not by 100 mM L-lysine. The results suggest that this protein is the arginine carrier.     

Regulation of carbamylphosphate synthesis in Serratia marcescens.

Serratia marcescens HY possessed a single carbamylphosphate synthase (CPSase) which was subject to cumulative repression by arginine and a pyrimidine. CPSase did not appear to be a part of a multifunctional enzyme complex as is the case for other enzymes of pyrimidine biosynthesis in this organism. CPSase was purified to homogeneity. The molecular weight of the enzyme was estimated to be 167,000 by sucrose density gradient ultracentrifugation. The double-reciprocal plot for magnesium adenosine triphosphate was linear, yielding a Km value of 2.5 mM. The enzyme utilized either glutamine (Km, 0.1 mM) or NH3 (Km, 10.5 mM) as a nitrogen donor in the reaction. CPSase activity was subject to activation by ornithine and feedback inhibition by uridine monophosphate, as is the case for other enteric bacteria. Carbamate kinase activity, detected in crude extracts of S. marcescens, was shown to be due to a constitutive acetate kinase. The absence of carbamate kinase from S. marcescens HY is consistent with the inability of this organism to utilize arginine as a source of energy under anaerobic conditions.     

Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria.

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.     

arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger.

In the absence of oxygen and nitrate, Pseudomonas aeruginosa metabolizes arginine via the arginine deiminase pathway, which allows slow growth on rich media. The conversion of arginine to ornithine, CO2, and NH3 is coupled to the production of ATP from ADP. The enzymes of the arginine deiminase pathway are organized in the arcDABC operon. The arcD gene encodes a hydrophobic polytopic membrane protein. Translocation of arginine and ornithine in membrane vesicles derived from an Escherichia coli strain harboring a recombinant plasmid carrying the arcD gene was studied. Arginine and ornithine uptake was coupled to the proton motive force with a bias toward the transmembrane electrical potential. Accumulated ornithine was readily exchangeable for external arginine or lysine. The exchange was several orders of magnitude faster than proton motive force-driven transport. The ArcD protein was reconstituted in proteoliposomes after detergent solubilization of membrane vesicles. These proteoliposomes mediate a stoichiometric exchange between arginine and ornithine. It is concluded that the ArcD protein is a transport system that catalyzes an electroneutral exchange between arginine and ornithine to allow high-efficiency energy conversion in the arginine deiminase pathway.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.     

Purification and properties of the arginine-specific carbamoyl-phosphate synthase from Saccharomyces cerevisiae.

The arginine-specific carbamoyl-phosphate synthase of yeast was stabilized sufficiently to allow partial purification of the enzyme (30- to 40-fold). The synthase (mol. wt 115000) comprised two unequal subunits: a heavy subunit (mol. wt 80000) capable of catalysing synthesis of carbamoyl phosphate with ammonia as a nitrogen donor and a light subunit conferring upon the holoenzyme the ability to utilize glutamine. The enzyme had unusually high affinity for ATP (Km = 0.2 mM) and atypical negative cooperativity for glutamine binding ([S]0.5 = 0.25 mM). Glutamine activity was not modulated by possible effectors such as arginine, ornithine or N-acetylglutamate. Thus, although the yeast arginine enzyme physically and functionally resembles the single enteric synthase, the systems differ substantially both in kinetic properties and in regulation of activity.