Pyrimidine synthesis, from aspartate

Orotic Acid. l,2,3,6.Tetrahydro-2,6-dioxo-4-py-nmidinecartroxyhc acid uracil-6-carboxylic acid whey factor animal galactose factor Oropur Orotyl. CjH.N.O, mol wt 156.10. C 38.47%. H 2.58%, N 17.95%. O 41.00%. A pyrimidine precursor in animal organisms, found in milk Bachstez. Ber. 64, 2683 (1931) Hilbert, J. Am. Chem. Soc. 54, 2082 (1932) Johnson, Schroeder, ibid. 2942. Synthesis from aspartic acid Nye. Mitchell, ibid. 69, 1382 (1947). Prepn by condensation of urea with monoethyl ester of ox -alacetic acid in methanol Scriabine, U.S. pat. 2,937,175... 

Pyrimidines are synthesized de novo in the cytoplasm from aspartate, COj, and glutamine as shown in Figure 1-18-2. Synthesis involves a cytoplasmic carbamoyl phosphate synthetase that differs from the mitochondrial enzyme with the same name used in the urea cycle. 

Biosynthesis of UMP. The parts of the intermediates derived from aspartate are shown in red. Bold type indicates atoms derived from carbamoyl phosphate. In contrast to purine nucleotide synthesis, where ring formation starts on the sugar, in pyrimidine biosynthesis the pyrimidine ring is completed before being attached to the ribose. 

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. 

Figure 25.2. de Novo Pathway for Pyrimidine Nucleotide Synthesis. The C-2 and N-3 atoms in the pyrimidine ring come from carbamoyl phosphate, whereas the other atoms of the ring come from aspartate. 

The answer is a. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) During purine ring biosynthesis, the amino acid glycine is completely incorporated to provide C4, C5, and N7. Glutamine contributes N3 and N9, aspartate provides Nl, and derivatives of tetrahydrofolate furnish C2 and C8. Carbon dioxide is the source of C6. In pyrimidine ring synthesis, C2 and N3 are derived from carbamoyl phosphate, while Nl, C4, C5, and C6 come from aspartate. 

Aspartate can be transaminated to form oxaloacetate, an intermediate of the citric-acid cycle. As with most transaminations, this is a reversible reaction, and aspartate can also be synthesized by a transamination reaction with glutamate and oxaloacetate to form aspartate and a-ketoglutarate. Therefore, aspartate is a nonessential amino acid. The aminotransferase with aspartate and a-ketoglutarate is particularly active in most tissues and occurs both in the mitochondria and the cytosol. The importance of this reaction is greater than simply forming the oxaloacetate or aspartate. Aspartate aminotransferase is an important reaction in the malate shuttle (see Chapter 11) wherein, reducing power can be transferred from the cytosol to the mitochondrion. Aspartate also plays a role in purine and pyrimidine synthesis and is particularly important in pyrimidine synthesis, where it donates both carbon and... 

Aspartate can be formed from oxaloacetate and glutamate, via transamination. This is important in urea synthesis, the malate shuttle, purine, and pyrimidine synthesis. 

The first step in the pathway, formation of carbamoyl aspartate from aspartate and carbamoyl phosphate, is the primary regulatory point in the pathway. The enzyme, aspartate transcarbamoylase (ATCase) (see here), is activated by ATP and inhibited by CTP, which is the end product of the pathway. Another point of regulation is CTP synthetase, which is feedback inhibited by CTP and activated by GTP. In bacteria, synthesis of ATCase subunits is inhibited by high levels of UTP. The inverted regulatory effects of purine and pyrimidines in the pathway are yet another way cells maintain a proper balance of nucleotides. 

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

Trichomonas vaginalis. T. vaginalis does not incorporate radiolabel from C-labeled aspartate, orotate or bicarbonate into its pyrimidines, suggesting the absence of de novo synthesis (76,77). Although there are conflicting reports as to the presence or absence of the specific enzymes of de novo synthesis (19,77,81), T. vaginalis appears to be incapable of de novo pyrimidine synthesis. 

Synthesis of the amino acids Eleven of the twenty common amino acids can be synthesized in the body (Fig. 39.1). The other nine are considered essential and must be obtained from the diet. Almost all of the amino acids that can be synthesized by humans are amino acids used for the synthesis of additional nitrogen-containing compounds. Examples include glycine, which is used for porphyrin and purine synthesis glutamate, which is required for neurotransmitter and purine synthesis and aspartate, which is required for both purine and pyrimidine biosynthesis. 

A different, simpler , pathway is involved in the synthesis of pyrimidine nucleotides. A pyrimidine base (orotate), is synthesised first. Then the ribose is added from 5-phosphoribosyl 1-pyrophosphate. The two precursors for the formation of orotate are carbamoylphosphate and aspartate, which form carbamoyl aspartate, catalysed by aspartate carbamoyltransferase. 

FIGURE 22-36 De novo synthesis of pyrimidine nucleotides biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphori-bosyltransferase. The first step in this pathway (not shown here see Fig. 18-11a) is the synthesis of carbamoyl phosphate from C02 and NH), catalyzed in eukaryotes by carbamoyl phosphate synthetase II. 

In de Novo Synthesis, the Pyrimidine Ring Is Assembled from Biearbonate, Aspartate, and Glutamine... 

In de novo synthesis of pyrimidines, the ring is synthesized first and then it is attached to ribose to form a pyrimidine nucleotide (Figure 25.2). Pyrimidine rings are assembled from bicarbonate, aspartic acid, and ammonia. Although ammonia can be used directly, it is usually produced from the hydrolysis of the side chain of glutamine. 

The pyrimidine ring is assembled first and then linked to ribose phosphate to form a pyrimidine nucleotide. PRPP is the donor of the ribose phosphate moiety. The synthesis of the pyrimidine ring starts with the formation of carbamoylaspartate from carbamoyl phosphate and aspartate, a reaction catalyzed by aspartate transcarbamoylase. Dehydration, cyclization, and oxidation yield orotate, which reacts with PRPP to give orotidylate. Decarboxylation of this pyrimidine nucleotide yields UMP. CTP is then formed by the amination of UTP. 

Making a pyrimidine. Write a balanced equation for the synthesis of orotate from glutamine, CO2, and aspartate. 

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