Orotic acid pyrimidine synthesis

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

Lieberman 1, A Komberg (1953) Enzymatic synthesis and breakdown of a pyrimidine, orotic acid I. Dihydro-orotic dehydrogenase. Biochim Biophys Acta 12 223-234. 

Urinary orotic acid generally is very elevated in babies with OTC deficiency and normal or even low in the infant with CPS deficiency. Patients with OTC deficiency have orotic aciduria because carbamyl phosphate spills into the cytoplasm, where it enters the pathway of pyrimidine synthesis. 

The two conditions can be distinguished by an increase in orotic add and uracil, which occurs in ornithine transcarbamoylase deficiency, but not in the defldency of carbamoyl phosphate synthetase. Orotic acid and uracil are intermediates in pyrimidine synthrais (see Chapter 18). This pathway is stimulated by the accumulation of carbamoyl phosphate, the substrate for ornithine transcarbamoylase in the urea cycle and for aspartate transcarbamoylase in pyrimidine synthesis. 

Answen B. Accumulation of orotic acid indicates megaloblastic anemia arises since pyrimidines are required for DNA synthesis. 

Carbamoyl phosphate synthetase formation in liver taken from tadpoles treated with thyroxine is enhanced by the addition of orotic acid, uracil or uridine (cytosine and adenosine had no effect). The synthesis of this enzyme is not affected by these pyrimidines in untreated animals. This indicates that there is a relative pyrimidine deficiency during thyroxine-induced metamorphosis [140]. 

The fact that many agents which interrupt the synthesis of pyrimidine nucleotides from orotic acid in animals can also inhibit the growth of experimental neoplasms suggests a search for additional antimetabolites whose locus of action is in this metabolic sequence. Two in vitro biological screening systems were developed for this purpose [202—207]. From a study of systems with oxidative energy sources, 5-bromo-[208—209] (Villa), 5-chloro-[210] (Vlllb) and 5-diazo-orotic acid [211] (IX) were found to inhibit the conversion of orotic acid to the uridine nucleotides by 40—100 per cent [202]. 

A common intermediate for all the nucleotides is 5-phosphoribosyl-l-diphosphate (PRPP), produced by successive ATP-dependent phosphorylations of ribose. This has an a-diphosphate leaving group that can be displaced in Sn2 reactions. Similar Sn2 reactions have been seen in glycoside synthesis (see Section 12.4) and biosynthesis (see Box 12.4), and for the synthesis of aminosugars (see Section 12.9). For pyrimidine nucleotide biosynthesis, the nucleophile is the 1-nitrogen of uracil-6-carboxylic acid, usually called orotic acid. The product is the nucleotide orotidylic acid, which is subsequently decarboxylated to the now recognizable uridylic acid (UMP). 

The second step in pyrimidine synthesis is the formation of car-bamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed hydrolytically by dihydroorotase. Thi resulting dihydroorotate is oxidized to produce orotic acid (onotate, Figure 22.21). The enzyme that produces orotate, dihydroorotate dehydrogenase, is located inside the mitochondria. All other reactions in pyrimidine biosynthesis are cytosolic. [Note The first three enzymes in this pathway (CPS II, aspartate transcarbamoylase, and dihydroorotase) are all domains of the same polypeptide chain. (See k p. 19 for a discussion of domains.) This is an example of a multifunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound.]... 

The end-product of pyrimidine base synthesis is orotic acid, which is converted to the nucleotide OMP by the addition of ribose 6-phosphate (donated by PRPP). OMP is then converted to UMP, which is phosphorylated to UTP. UTP is then aminated to form CTP. A deficiency of the enzyme complex (UMP synthase) that converts orotic acid to UMP causes orotic aciduria. 

Vitamins and Minerals. Milk is a rich source of vitamins and other organic substances that stimulate microbial growth. Niacin, biotin, and pantothenic acid are required for growth by lactic streptococci (Reiter and Oram 1962). Thus the presence of an ample quantity of B-complex vitamins makes milk an excellent growth medium for these and other lactic acid bacteria. Milk is also a good source of orotic acid, a metabolic precursor of the pyrimidines required for nucleic acid synthesis. Fermentation can either increase or decrease the vitamin content of milk products (Deeth and Tamime 1981 Reddy et al. 1976). The folic acid and vitamin Bi2 content of cultured milk depends on the species and strain of culture used and the incubation conditions (Rao et al. 1984). When mixed cultures are used, excretion of B-complex vita-... 

Eukaryotic organisms contain a multifunctional enzyme with carbamoylphosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activities. Two mechanisms control this enzyme. First, control at the level of enzyme synthesis exists the transcription of the gene for the enzyme is reduced if an excess of pyrimidines is present. Secondly, control exists at the level of feedback inhibition by pyrimidine nucleotides. This enzyme is also an example of the phenomenon of metabolic channeling aspartate, ammonia, and carbon dioxide enter the enzyme and come out as orotic acid. 

Orotic acid is an intermediate in pyrimidine synthesis. It is synthesized from the transcar-bamylation of aspartic acid and subsequent intramolecular condensation. Any defect in ureagenesis causing accumulation of intracellular carbamoyl phosphate provides substrate for orotic acid synthesis. Therefore, a defect of OTC, or any defect distal to this step, can cause orotic aciduria. The detection of elevated orotic acid in the urine is most useful in differentiating between patients with OTC deficiency and either CPSI- or NAGS-deficient patients in whom orotic aciduria is not present. 

A second, cytosolic CPS activity (CPSII) occurs in mammals as part of the CAD trifunctional protein that catalyzes the first three steps of pyrimidine synthesis (CPSII, asparate tran-scarbamoylase, and dihydroorotase). The activities of these three enzymes—CPSII, aspartate transcarbamoylase, and dihydroorotase—result in the production of orotic acid from ammonium, bicarbonate, and ATP. CPSII has no role in ureagenesis, but orotic aciduria results from hepatocellular accumulation of carbamyl phosphate and helps distinguish CPSI deficiency from other UCDs. Defects in CPSI classically present with neonatal acute hyperammonemic encephalopathy. The plasma citrulline and urine orotic acid concentrations are both low. A definitive diagnosis can be established by enzyme assay of biopsied liver tissue or by mutation analysis. 

The fourth step in the de novo synthesis of pyrimidine nucleotides—the conversion of dihydroorotic acid to orotic acid—is catalyzed by dihydroorotic acid dehydrogenase. The enzyme, located on the cytosolic side of the inner membrane of mitochondria, is a target for antitumor agents. 

B o A factor for feather growth in chickens, probably folic acid and thiamin B Later identified as a mixture of folic acid and thiamin B 3 a growth factor in rats orotic acid, intermediate in pyrimidine synthesis... 

B-8) Orotic aciduria. Blocks in steps leading to pyrimidine synthesis may result in deficient production of pyrimidine nucleotides. There may be anemia and immune deficiency (from decreased red and while cell production) and excess orotic acid which may precipitate in the urine. Treatment with uridine may not only help supply the missing pyrimidine, but can decrease the level of orotate by uridine s negative feedback on steps that lead to orotate production. 

Treatment involves a low-protein diet (0.5-0.7 g/kg BW/day) with a sufficient supply of calories. Substitution of essential amino acids (in about the same quantity) is required. The administration of benzoate (0.1-0.25 g/kg BW/day), arginine hydrochloride (1 mmol/kg BW/day) or sodium phenylacetate (0.3-0.5 g/ kg BW/day) (phenylbutyrate tends to be more effective) facilitates nitrogen excretion via other metabolic pathways. (168-170) With an enhanced excretion of orotate or other metabolites of pyrimidine synthesis, the administration of allopurinol leads to an increase in the excretion of nitrogen via metabolites from pyrimidine synthesis. Ammonia and urea precursors are eliminated by haemodialysis. In individual cases, liver transplantation is indicated. (I7l)... 

Brequinar (DUP 785, NSC 368390) is a quinoline carboxylic acid derivative that inhibits pyrimidine synthesis by inhibiting dihydro-orotate dehydrogenase. It was originally developed as an anticancer drug, but has also been investigated for its immunosuppressant activity after transplantation. Some data suggest that that the immunosuppressant activity of brequinar may be partly due to inhibition of tyrosine phosphorylation in lymphocytes (1). 

The first evidence of the effect of elevation of blood ammonia on the synthesis and breakdown of pyrimidine was found in cases of hyperammonemia (L8), presumably because it is in this condition that the highest levels of blood ammonia are likely to be found. Orotic acid was first detected in the urine of these patients, because on one occasion so... 

In the first case of citrullinemia reported (M6), intermediary metabolites of the pyrimidine pathway were not sought in the urine. In the second, orotic acid was tested for, but not detected (M12). In the third child, orotic acid, uridine, and uracil were found in relatively large amounts in the urine when the plasma glutamine was at the very high level of 41.0 mg/100 ml, showing that in this genetic disorder, as in the other disorders of urea synthesis, these metabolites are always excreted in excess when the glutamine is raised (VI). 

Immune system dysfunction in ADA deficiency has also been ascribed to the inhibition of pyrimidine nucleotide synthesis by adenosine, known as pyrimidine starvation. This may arise from inhibition of conversion of orotic acid to orotidine 5 -monophosphate or from inhibition of PRPP synthesis by excessive synthesis of adenine nucleotides. 

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