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The De Novo Biosynthesis of Uridine Monophosphate (UMP) Involves Six Enzymatic Reactions and Appears to Be Encoded by Only Three Structural Genes in Animals

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The De Novo Biosynthesis of Uridine Monophosphate (UMP) Involves Six Enzymatic Reactions and Appears to Be Encoded by Only Three Structural Genes in Animals J. Nutr. Sci. Vitaminol., 37, 517-528, 1991 Effect of Dietary Protein on Pyrimidine-Metabolizing Enzymes in Rats Masae KANEKO, Shigeko FUJIMOTO, Mariko KIKUGAWA, Yasuhide KONTANI, and Nanaya TAMAKI* Laboratory of Nutritional Chemistry, Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe 651-21, Japan (Received February 25, 1991) Summary The effect of dietary protein on pyrimidine-metabolizing enzymes was studied in the rat. The activities of dihydropyrimidine dehydrogenase and ƒÀ-ureidopropionase in the livers of rats fed a protein free diet were significantly decreased, while the activity of dihydropyrim idinase was unaffected. Protein deficiency (5%) also decreased the activity of ƒÀ-ureidopropionase. On the other hand, a high-protein diet (60%) increased the level of ƒÀ-ureidopropionase. The activities of ƒÀ- alanine-oxoglutarate aminotransferase (aminobutyrate aminotransferase) and D-3-aminoisobutyrate-pyruvate aminotransferase ((R)-3-amino-2- methylpropionate-pyruvate aminotransferase), which are present in mi tochondria, depended on the amount of protein in the diet. Ammonium ions supplemented in the diet and given by injection did not affect the activities of rat liver pyrimidine-metabolizing enzymes (dihydropyrimi dine dehydrogenase, dihydropyrimidinase, ƒÀ-ureidopropionase, ƒÀ-alanine - oxoglutarate aminotransferase and D-3-aminoisobutyrate-pyruvate ami notransferase). Dietary uridine resulted in the accumulation of uracil in the liver, but did not affect the activities of pyrimidine-metabolizing enzymes. Key Words dihydropyrimidine dehydrogenase, dihydropyrimidinase, ƒÀ- ureidopropionase, ƒÀ-alanine-oxoglutarate aminotransferase, D-3-amino isobutyrate-pyruvate aminotransferase, pyrimidine The de novo biosynthesis of uridine monophosphate (UMP) involves six enzymatic reactions and appears to be encoded by only three structural genes in animals. The active sites of the first three enzymes, carbamoyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase, are on a single large polypeptide * To whom correspondence should be addressed . Abbreviations: ƒÀ-AlaAT I, ƒÀ-alanine-oxoglutarate aminotransferase; ƒÀ-AlaAT II, D-3 - aminoisobutyrate-pyruvate aminotransferase. 517 518 M. KANEKO et al. that aggregates to form the native multienzymatic protein (1-3) . The active sites of the fifth and sixth enzymes of the pathway , orotate phosphoribosyl-transferase and orotidylate decarboxylase, are also composed of a single polypeptide chain (4) . Carbamoyl-phosphate synthetase is the rate-limiting enzyme in the de novo UMP - biosynthetic pathway and is the site of feedback inhibition by uridine triphosphate (UTP) and activation by 5-phosphoribose 1-bisphosphate (5, 6). Weber et al. (7) found that the incorporation of thymidine into DNA inceased after partial hep atectomy, and with hepatoma growth, but decreased in rat livers over time after birth. On the other hand, the catabolism of thymidine to CO2 increased with post-natal time and was inactivated after partial hepatectomy and in rapidly growing neoplasms (7). Uracil and thymine are metabolized to ƒÀ-alanine and ƒÀ- aminoisobutyrate, respectively, by dihydropyrimidine dehydrogenase , dihydropy rimidinase and ƒÀ-ureidopropionase in cytosol , as ilustrated in Fig. 1. ƒÀ-Alanine and ƒÀ-aminoisobutyrate are transported into mitochondria (8 , 9), where they are further metabolized to acetyl-CoA and propionyl-CoA , respectively, by ƒÀ-alanine - oxoglutarate aminotransferase (ƒÀ-AlaAT I), D-3-aminoisobutyrate-pyruvate ami notransferse (ƒÀ-AlaAT II) and methylmalonate semialdehyde dehydrogenase in the mitochondrial matrix (9-11). Dihydropyrimidine dehydrogenase has been Fig. 1. Degradation pathway of pyrimidine. 1, dihydropyrimidine dehydrogenase [EC 1.3.1.1]; 2, dihydropyrimidinase [EC 3.5.2.2]; 3, ƒÀ-ureidopropionase [EC 3.5.1.6]; 4, aminobutyrate aminotransferase [EC 2.6 .1.19]; 5, (R)-3-amino-2 - methylpropionate-pyruvate aminotransferase [EC 2 .6.1.40]; 6, methylmalonate semialdehyde dehydrogenase [EC 1.2.1.27]. Abbreviations: ƒÀ-Ala , ƒÀ-alanine; ƒÀ- AIB, ƒÀ-aminoisobutyrate. J Nutr. Sci. Vitaminol. PROTEIN LEVEL ON PYRIMIDINE METABOLISM 519 identified as the rate-limiting enzyme of pyrimidine catabolism (12, 13). In the spectrum of hepatomas, dihydropyrimidine dehydrogenase activity decreased in parallel with increased growth rate (7, 14). In the livers of rats fed a protein-deficient diet, DNA synthesis and activity of thymidine kinase decreased to less than half of control values (15). In contrast, incorporation of [6-14C]-orotate into RNA and uridine kinase activity significantly increased (15). Ammonium ions have been shown to stimulate pyrimidine bio synthesis as a result of carbamoyl phosphate synthesis by mitochondria carbamoyl phosphate synthetase (16-18). In order to define the relevance of normal and abnormally increased uracil for hepatic pyrimidine catabolism, we investigated the effect of dietary protein, ammonium ions and uridine on pyrimidine-catabolizing enzymes in the rat liver. MATERIALS AND METHODS Chemicals. All chemicals used were of analytical grade and were purchased from Nacalai Tesque (Kyoto) unless otherwise stated. Uridine was a product of Sigma Chemicals. 5-Bromo-5,6-dihydrouracil was synthesized from 5,6-dihydro uracil by direct bromination (19). ƒÀ-[2-14C]alanine was purchased from New England Nuclear. Materials for the animals' diets were obtained from Oriental Yeast Ltd., Tokyo. Animals. Male albino rats (Sprague-Dawley, 130-150g) were housed in individual screen-bottom cages in a room maintained at 23•}1•Ž with 50% hu midity under controlled lighting conditions (12h light-dark cycle). The animals were fed a commercial stock diet and water ad libitum for 1 week before the experiment to acclimatize them to the new environment. Acclimatized rats showing progressive weight gain were selected and separated into groups. All animals were sacrificed between 10:00a.m. and noon except those used in the ammonium acetate injection experiments. Low and high protein diets. The compositions of the low and high-protein diets are shown in Table 1. The animals received 20g of feed per day. Ammonium chloride and uridine diets, and ammonium acetate injection. The composition of ammonium chloride and uridine diets is shown in Table 2. The 20% casein diet was used as the control. Ammonium acetate was dissolved in physiological saline to be prepared to 3.7 M and 3.7mol/kg of body weight was intraperitoneally injected 4 or 8 times at 1-h intervals. One hour after the last injection, the animals were sacrificed. Enzyme assays: dihydropyrimidine dehydrogenase. The livers were homog enized in 10vol. buffer A (10mM potassium phosphate, pH 7.4, containing 5mM 2 - mercaptoethanol and 2.5mM MgCl2). After centrifugation, the supernatant was heated to 50•Ž for 1min, and then cooled to 4•Ž. The precipitate was discarded after centrifugation and the supernatant was adjusted to pH 4.85 with 5% acetate. After centrifugation, the supernatant was neutralized with 0.5M KOH and treated Vol. 37, No. 5, 1991 520 M. KANEKO et al. Table 1. Compositions of the experimental diets . a Sucrose e corn starch=2 :1. b Minerals were (in g/kg of diet): CaHPO4•E2H2O 8 .736; KH2PO4 15.432; NaH2PO4 5,610; NaCl 2 .796; Ca-lactate 21,054; Fe-citrate 1908; MgSO4 4.302; ZnCO3 0.066; MnSO4•E4-6H2O 0 .072; CuSO4•E5H2O 0.018; KI 0.006. c Vitamins were (in IU/kg of diet); retinyl acetate 10 ,000; cholecalciferol 2,000, and (in mg/kg diet): ƒ¿-tocopheryl acetate 100; menadione 104; thiamin hydrochloride 24; riboflavin 80; pyridoxine hydrochloride 16; cyanocobalamin 0 .01; ascorbic acid 600; D-biotin 0.4; folic acid 4; calcium pantothenate 100; p-aminobenzoic acid 100; niacin 120; choline chloride 4,000. Table 2. Compositions of the diets supplemented with ammonium chloride and uridine. 1 Diets used for the experiments on the effect of ammonium ions . 2 Diets used for the experiments on the effect of uridine . a Sucrose: corn starch=2:1. b Salt mixture and vitamin mixture are shown in Table 1. with ammonium sulfate. The precipitate obtained at 30-50% saturation was dissolved in a minimum volume of buffer A and used for enzyme analysis . Dihydropyrimidine dehydrogenase activity was followed by measuring the rate of the disappearance of NADPH at 37•Ž (20) . The standard assay mixture contained 50mM potassium phosphate, pH 7 .4, including 0.15mM uracil and 0.15 mM NADPH in a total volume of 3 .0ml. Dihydropyrimidinase. The livers were homogenized in 10vol . 10mM potas sium phosphate, pH 7.0, containing 10mM 2-mercaptoethanol . After centrifu gation, the supernatant was used for analyses of dihydropyrimidinase and ,ƒÀ- J. Nutr. Sci. Vitaminol. PROTEIN LEVEL ON PYRIMIDINE METABOLISM 521 ureidopropionase activities. Dihydropyrimidinase was measured by the method of Brooks et al. (21). The enzyme activity was assessed by measuring the rate of disappearance of 5-bromo - 5,6-dihydrouracil at 225nm in a cuvette with a 1.0cm light path at 37•Ž. The extinction coefficient of 5-bromo-5,6-dihydrouracil at 225nm was 3,24•~103M-1•E cm-1, at pH 8.2, in 50mM Tris-HCl buffer pH 8.2 and 0.17mM 5-bromo-5,6 - dihydrouracil in a total volume of 3.0ml. ƒÀ-Ureidopropionase . ƒÀ-Ureidopropionase activity was measured with respect to the rate of formation of ammonia (22). The standard reaction mixture con tained 0.1M sodium phosphate, pH 7.0, including bovine serum albumin (0.1%), 10mM MgCl2, 1mM EDTA, 5mM 2-mercaptoethanol, and 2mM N-carbamoyl-ƒÀ- alanine. Incubation was carried out in a shaking water bath for 30min at 37•Ž.ƒÀ -AlaAT I . The livers were homogenized in 10vol. 10mM potassium phos phate, pH 7.5, containing 1mM EDTA, 2mM 2-mercaptoethanol and 40ƒÊM pyridoxal 5•L-phosphate. The homogenate was briefly centrifuged. The supernatant was used to analyze ƒÀ-AlaAT I and ƒÀ-AlaAT II activities. The activity of ƒÀ-AlaAT I was determined by the amount of malonate semialdehyde produced from ƒÀ-alanine with 2-oxoglutarate according to methods previously described (23). The reaction mixture contained 50mM sodium borate (pH 8.8), 5mM 2-mercaptoethanol, 0.5mM pyridoxal 5•L-phosphate, 1mM ƒÀ- [2-14C]alanine (specific activity 37 GBq/mol) and l0mM 2-oxoglutarate in a final volume of 1.0ml.
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