Cytosol pyrimidine synthesis

The carbamoyl phosphate synthetase (abbreviated to CPS-I) that is involved in the ornithine cycle differs from the enzyme that is involved in pyrimidine synthesis (carbamoyl phosphate synthetase-ll). The latter enzyme is cytosolic, requires glutamine for provision of nitrogen, rather than ammonia, and is regulated by different factors (Chapter 20). 

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 design for pyrimidine synthesis differs somewhat from that of purine biosynthesis in that the sugar is attached to the pyrimidine ring at the end of the pathway. In addition, pyrimidine biosynthesis occurs in part in the cytosol and in part in the mitochondria and involves the participation of two multifunctional enzymes. The pathway is summarized in Figure 10.9. One of the initial reactants is the compound carbamoyl phosphate (carbamoyl phosphoric acid). This compound is also formed in the urea biosynthetic pathway, but this takes place in the mitochondria and requires NH3 (Chapter 20). The cytosolic biosynthesis of carbamoyl phosphate for the purpose of pyrimidine biosynthesis requires glutamine as the nitrogen donor ... 

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. 

In the first reaction, glutamine reacts with C02 and 2 ATP to form carbamoyl phosphate. This reaction is analogous to the first reaction of the urea cycle. However, for pyrimidine synthesis, glutamine provides the nitrogen and the reaction occurs in the cytosol, where it is catalyzed by carbamoyl phosphate synthetase II, which is inhibited by UTP. 

Deficiency of folate or vitamin Bn can cause hematological changes similar to hereditary orotic aciduria. Folate is directly involved in thymidylic acid synthesis and indirectly involved in vitamin Bn synthesis. Orotic aciduria without the characteristic hematological abnormalities occurs in disorders of the urea cycle that lead to accumulation of carbamoyl phosphate in mitochondria (e.g., ornithine transcarbamoylase deficiency see Chapter 17). The carbamoyl phosphate exits from the mitochondria and augments cytosolic pyrimidine biosynthesis. Treatment with allopurinol or 6-azauridine also produces orotic aciduria as a result of inhibition of orotidine-5 phosphate decarboxylase by their metabolic products. 

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... 

Dihydro-orotate-oxidizing activity in rat liver homogenates can be recovered completely in the mitochondrial fraction [103,104].With the exception of this system all the other enzymes of the orotate pathway appear to be present in the soluble cytosolic fraction. Dihydro-orotate dehydrogenase from rat liver was found to be located on the outer surface of the inner membrane of mitochondria [105]. Dihydro-orotate can diffuse freely from the cytosol into the mitochondria and orotate can diffuse freely from the mitochondria into the cytosol. Therefore no active transport of either dihydro-orotate or orotate is required in pyrimidine synthesis [105]. In addition to inhibiting dihydro-orotase, orotic acid strongly blocks [103] dihydro-orotate oxidation. 

Figure 34-7 summarizes the roles of the intermediates and enzymes of pyrimidine nucleotide biosynthesis. The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthase II, a different enzyme from the mitochondrial carbamoyl phosphate synthase I of urea synthesis (Figure 29-9). Compartmentation thus provides two independent pools of carbamoyl phosphate. PRPP, an early participant in purine nucleotide synthesis (Figure 34-2), is a much later participant in pyrimidine biosynthesis.

Cytosol Glycolysis, glycogenesis and glycogenolysis, hexose monophosphate pathway, fatty acid synthesis, purine and pyrimidine catabolism, aminoacyl-tRNA synthetases... 

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. 

In humans, there are two immunologically distinct carbamoyl phosphate synthases, one mitochondrial (CPSI) and the other cytosolic (CPSII). CPSI is involved in ure-agenesis, uses NH3 exclusively as the nitrogen donor, and requires binding of NAG for activity. CPSII uses glutamine as substrate, is not dependent on NAG for activity, and is required for synthesis of pyrimidine... 

The metabolic interrelationship between mitochondrial carbamoyl phosphate synthesis to urea formation and to cytosolic carbamoyl phosphate channeled into pyrimidine biosynthesis. In ornithine transcarbamoyiase (OTC) deficiency, mitochondrial carbamoyl phosphate diffuses into the cytosol and stimulates pyrimidine biosynthesis, leading to orotidinuria. Administration of allopurinol augments orotidinuria by increasing the flux in the pyrimidine biosynthetic pathway. CPS = Carbamoyl phosphate synthase, AT = aspartate transcarbamoyiase, D = dihydroorotase, DH = dihydroorotate dehydrogenase, OPRT = orotate phosphoribosyltransferase, XO = xanthine oxida.se,... 

DHODs are FMN-containing enzymes that convert dihydroorotate (DHO) to orotate (OA) in the only redox step in the wow synthesis of pyrimidines (Scheme 12). DHODs have been grouped into two classes based on sequence. Class 2 DHODs are membrane-bound monomers that are reoxidized by ubiquinone, coupling pyrimidine biosynthesis to the respiratory chain. On the other hand, Class 1 DHODs are cytosolic proteins that have been further divided into two subclasses. Class lA DHODs are homodimers that are reoxidized by fumarate. Class 1B DHODs are azfiz heterotetramers with an FMN-containing subunit very similar to Class 1A enzymes and a second subunit that contains an iron—sulfur cluster and FAD, allowing Class IB DHODs to be reoxidized by NAD. ... 

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