BEAT WIPFt AND THOMAS LEISINGER*. Mikrobiologisches Institut, Eidgenossische .... method of Lowry et al. (11) with bovine serum albumin as a standard.
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 874-880
Vol. 140, No. 3
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Regulation of Activity and Synthesis of N-Acetylglutamate Synthase from Saccharomyces cerevisiae BEAT WIPFt AND THOMAS LEISINGER* Mikrobiologisches Institut, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zurich, Switzerland Received for publication 18 September 1979
Feedback inhibition of N-acetylglutamate synthase in a particulate fraction from Saccharomyces cerevisiae by L-arginine was synergistically enhanced by Nactylglutamate, whereas coenzyme A led to an additive enhancement of arginine inhibition. N-acetylglutamate synthase was not inhibited by polyamines, nor was the enzyme inactivated by incubation in the presence of coenzyme A and zinc ions. Evidence was obtained for the involvement of at least three different regulatory mechanisms in the expression of N-acetylglutamate synthase: argininespecific repression, glucose repression and general amino acid control. The combined action of these control mechanisms led to a 90-fold variation in the specific activity of the enzyme. The transacetylase modification of ornithine biosynthesis (21) has been detected in fungi (2), in algae (4, 16), and in several bacteria like Micrococcus (21), Pseudomonas (21), and photosynthetic bacteria (8). This modification of the pathway provides two enzymes for the N-acetylation of L-glutamic acid, the first step in ornithine biosynthesis: N-acetylglutamate synthase (acetyl coenzyme A [acetyl-CoA]:L-glutamate N-acetyltransferase, EC 2.3.1.1) and ornithine acetyltransferase (N2-acetyl-L-ornithine:L-glutamate N-acetyltransferase, EC 2.3.1.35). N-acetylglutamate synthase catalyzes an acetyl-CoAconsuming reaction and, by replenishing the pool of acetylated intermediates of ornithine biosynthesis, fulfills an anaplerotic function. Ornithine acetyltransferase generates N-acetylglutamate without expenditure of energy and is responsible for the cyclic mode of ornithine synthesis. In Saccharomyces cerevisiae the enzymes of ornithine biosynthesis are located in the mitochondria (9, 26). Mutations in argA or argE, the structural genes of N-acetylglutamate synthase and ornithine acetyltransferase, respectively, lead to an ornithine requirement in this organism (2, 23). Extensive studies on the regulation of arginine biosynthesis in S. cerevisiae by Wiame and collaborators have revealed the following mechanisms governing activity and synthesis of the eight arginine biosynthetic enzymes: (i) feedback inhibition by arginine of N-acetylglutamate 5phosphotransferase, the second enzyme of the pathway (1, 7); (ii) epiarginasic control inacti-
vating ornithine carbamoyltransferase by the formation of a complex with arginase in the presence of high intracellular concentrations of arginine and ornithine (15, 18, 24); (iii) argininespecific repression regulating the synthesis of at least five of the eight biosynthetic enzymes (23); (iv) General amino acid control affecting the expression of arginine biosynthetic enzymes together with enzymes belonging to the biosynthesis of histidine, lysine, and the aliphatic and the aromatic amino acids (3, 13, 14, 17, 27). This mechanism has been shown to occur under nitrogen-limited growth as well as under nitrogen excess (17). N-acetylglutamate synthase was not considered in these studies, because until recently the enzyme could not be detected in crude extracts. Since an assay method for this enzyme is now available (9, 26), we report in the present communication on a survey of the regulatory effects controlling activity and expression of N-acetylglutamate synthase. For comparison we have included data on the regulation of the synthesis of ornithine acetyltransferase, an enzyme not subject to arginine-specific repression (23).
t Present address: Hoffmann-La Roche & Co. AG, CH-4058 Basel, Switzerland. 874
MATERIALS AND METHODS Strains. The wild-type strain X2180-A of S. cerevisiae, originally obtained from T. R. Manney, Manhattan, Kan., was used in this study. Strain RH 657 is a leucine bradytrophic mutant derived from strain X2180-A by P. Niederberger. Media and culture conditions. Cells were gpown on a minimal medium containing 1% glucose, 0.145% yeast nitrogen base lacking amino acids and ammonium sulphate (Difco), and 0.525% ammonium sulphate. When the organism was grown in Erlenmeyer
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flasks equipped with baffles, a buffer consisting of 1% citric acid and 0.64% potassium hydroxide, pH 5.0, was included in the medium. Cultures in flasks were incubated at 30°C on a rotatory shaker at 80 rpm. Experiments on the regulation of enzyme synthesis were done with 10-liter cultures grown in a 14-liter P.E.C. GF-0014 fermentor (Chemap AG, Mannedorf, Switzerland). Medium for these cultures did not contain buffer, and the concentration of yeast nitrogen base was raised to 0.29%. The fermentor was equipped with temperature and pH control, a rotor blade stirrer, and baffles. Stirring was set at 900 rpm, temperature was adjusted at 300C, and pH was controlled at 5.0 by automatic addition of 4 M sodium hydroxide. Aeration was set to 0.1 vvm (volume of air per volume of medium per minute) after inoculation and raised stepwise to 1.0 vvm before the culture reached the diauxic lag phase of growth. L-Arginine * hydrochloride was added to the medium at 1 mg/mi. Nonlimited growth of strain RH 657 was achieved by the addition of 0.5 mg of L-leucine and 0.5 mg of L-valine per ml of medium. Valine was necessary because high concentrations of leucine led to valine limitation in strain RH 657 (17). Addition of 0.04 mg of each amino acid per ml allowed nonlimited growth of the mutant up to about 0.6 g (dry weight) per liter. Media in Erlenmeyer flasks were inoculated with exponentially growing cells adapted to the same medium. The fermentor was inoculated with 200 ml of an exponentially growing flask culture. The dry weight of cells was determined by filtration of a sample of a culture through a glass fiber filter (Whatman GF/C, 2.4-cm diameter). The filters were rinsed with distilled water and dried at 70°C to constant weight. Preparation of cell extracts. Enzyme activities were determined either in crude cell extracts or in particulate fractions. For the preparation of crude cell extracts, washed cells were suspended in 10 mM potassium phosphate1 mM EDTA, pH 7.0 (0.25 g [wet weight] of cells per ml of buffer) and broken by treatment with a French pressure cell at a pressure of 60,000 kPa. Whole cells and cell debris were removed by centrifugation (800 x g for 10 min), and the supernatant yielded the crude cell extract. It was dialyzed overnight against three changes of the same buffer and stored at -20°C. For the preparation of particulate fractions, cells were suspended in 10 mM potassium phosphate (pH 7.0)-i mM EDTA-0.5 M D-sorbitol (0.3 g [wet weight] of cells per ml of buffer) and mixed with an equal volume of glass beads (0.45-mm diameter). The suspension was agitated with a Vibromixer (Chemap AG, Mannedorf, Switzerland) for 2 min. The cell suspension was separated from the glass beads and centrifuged for 10 min at 1,000 x g. The supernatant was decanted and centrifuged again at 12,000 x g for 45 min. The sediment was resuspended in the same buffer, centrifuged, and finally suspended in 10 mM potassium phosphate (pH 7.0)-i mM EDTA. The particulate fraction thus obtained was dialyzed and stored at -20°C. The specific activity of N-acetylglutamate synthase was about fourfold higher in a particulate fraction than in a crude extract. Protein assay. Protein was determined by the
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method of Lowry et al. (11) with bovine serum albumin as a standard. Enzyme assays. N-acetylglutamate synthase was assayed by the procedure of Haas et al. (5) with the following modification. The standard incubation mixture contained in a volume of 50 ul: 200 mM Trishydrochloride (pH 9.0), 7 mM amino-oxiacetic acid, 20 mM L-[U-'4C]glutamic acid (180,uCi/mmol), 5 mM acetyl-CoA, and protein (10 to 100 ,ug). Blank values were obtained by including 1 mM L-arginine in the incubation mixture. All the reagents were mixed at 00C, and the reaction was started by the addition of enzyme and subsequent incubation at 30°C. After 10 min, the reaction was stopped by the addition of 0.1 ml of 0.3 M HCI. A 0.1-ml amount of the mixture was applied to a Dowex 5OW column (bed volume, 0.5 by 2.5 cm), and N-acetylglutamate was eluted with two 0.5-ml portions of 0.1 N HCl. The eluate was collected in a scintillation vial and mixed with 7.5 ml of scintillation cocktail. Ornithine acetyltransferase was assayed by the same procedure except that acetyl-CoA was replaced by 5 mM N2-acetyl-L-ornithine. For both enzymes one unit of enzymatic activity is defined as the amount of enzyme catalyzing the formation of 1 nmol of N-acetylglutamate per min under standard assay conditions. Chemicals. L-[U-'4C]glutamic acid was purchased from New England Nuclear Corp., Boston, Mass. It was purified before use by ion-exchange chromatography on Dowex 50W. Acetyl-CoA and N2-acetyl-Lornithine were obtained from Boehringer, Mannheim, Germany. Dowex 5OW, X8, 200 to 400 mesh, H+ form, was obtained from Fluka AG, Buchs SG, Switzerland. All other chemicals, amino acids, etc., were purchased either from Fluka AG or from Merck, Darmstadt, Germany. Scintillation cocktail was mixed from 2 volumes of toluene containing 4 g of PPO (2,5-diphenyloxazole) and 0.1 g of POPOP [1,4-bis-(5-phenyloxazolyl)benzene] per liter and 1 volume of Triton X-100.
RESULTS Regulation of enzyme activity. In crude cell extracts N-acetylglutamate synthase was 10 to 15% inhibited by sulfhydryl reagents. No requirements for metal ions could be detected. The presence of 10 mM magnesium chloride in the assay reduced the activity of the enzyme by 10%. The amount of product formed increased linearly with time for at least 30 min and was proportional to the protein concentration between 10 and 100 ,ug of protein per assay. The velocity of the enzyme reaction was maximal at pH values between 8.5 and 9.5. At pH values below 8.5, it decreased sharply. Figure 1 shows the strong inhibition of enzyme activity by L-arginine. Under standard assay conditions 0.02 mM L-arginine inhibited the enzyme by 50%. At 0.1 mM L-arginine, enzyme activity was completely suppressed. Experiments on the effect of additional effectors on the activity of N-acetylglutamate syn-
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