Glutamine pyrimidine biosynthesi

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthase I (reaction 1, Figure 29-9). A cytosolic form of this enzyme, carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Chapter 34). Carbamoyl phosphate synthase I, the rate-hmiting enzyme of the urea cycle, is active only in the presence of its allosteric activator JV-acetylglutamate, which enhances the affinity of the synthase for ATP. Formation of carbamoyl phosphate requires 2 mol of ATP, one of which serves as a phosphate donor. Conversion of the second ATP to AMP and pyrophosphate, coupled to the hydrolysis of pyrophosphate to orthophosphate, provides the driving... 

In eukaryotic cells, the two enzymes are in different cellular compartments. Form I uses ammonia and is mitochondrial its function is to provide activated ammonia for arginine biosynthesis (and urea formation during Nitrogen elimination). Form II uses glutamine and is cytoplasmic it functions in pyrimidine biosynthesis. 

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 ... 

Dihydroorotate dehydrogenase, the enzyme catalyzing the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 15-15), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group and in mammals electrons are passed to ubiquinone. The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol must pass across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol to be a substrate for bifunctional UMP synthase. Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD, and an ammonia-dependent enzyme (CPSase /) which is found in the mitochondrial matrix, and which is used for urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytosol. 

Pyrimidine biosynthesis in E. coli is regulated by the feedback inhibition of aspartate transcarbamoylase, the enzyme that catalyzes the committed step. CTP inhibits and ATP stimulates this enzyme. The feedback inhibition of glutamine-PRPP amidotransferase by purine nucleotides is important in regulating their biosynthesis. 

FIGURE 9.7 Pyrimidine biosynthetic pathway. The pathway of pyrimidine biosynthesis involves sbc steps and results in the production of uridine 58-monophosphate. Folate is not used in this pathway The pathway commences with the transfer of the amide nitrogen of glutamine to bicarbonate to produce caibamy I phosphate. This molecule then reacts with aspartate to form the beginnings of the siK-membered pyrimidine ring. 

C. Options A and B are true for purine but not pyrimidine biosynthesis. During pyrimidine synthesis, the entire aspartate molecule is incorporated into the ring. Glutamine is the substrate for carbamoyl phosphate synthetase II, the enzyme involved in pyrimidine biosynthesis. (NH4+ is the substrate for synthetase I used in urea synthesis.) Glydne supplies one nitrogen for purine synthesis. 

C. In pyrimidine biosynthesis, carbamoyl phosphate, produced from glutamine, C02, and ATP, reacts with aspartate to form a base which, after oxidation, reacts with phosphoribosyl... 

T2. Tatibana, M., and Ito, K., Control of pyrimidine biosynthesis in mammalian tissues. 1. Partial purification and characterization of glutamine utilising carbamyl phosphate synthetase of mouse spleen and its tissue distribution. J. Biol. Chem. 244, 5403-5413 (1969). 

In eukaryotic cells, two separate pools of carbamoyl phosphate are synthesized by different enzymes located at different sites. Carbamoyl phosphate synthetase I (CPS I) is located in the inner membrane of mitochondria in the liver and, to lesser extent, in the kidneys and small intestine. It supplies carbamoyl phosphate for the urea cycle. CPS 1 is specific for ammonia as nitrogen donor and requires N-acetylglutamate as activator. Carbamoyl phosphate synthetase II (CPS II) is present in the cytosol. It supplies carbamoyl phosphate for pyrimidine nucleotide biosynthesis and uses the amido group of glutamine as nitrogen donor. The presence of physically separated CPSs in eukaryotes probably reflects the need for independent regulation of pyrimidine biosynthesis and urea formation, despite the fact that both pathways require carbamoyl phosphate. In prokaryotes, one CPS serves both pathways. 

The starting materials for the pyrimidine biosynthesis are aspartic acid (14) and carbamoyl phosphoric acid (17) the latter is synthesized alongwith glutamic acid (16) from ATP and glutamine (15) as shown below ... 

Although the GOGAT reaction consumes one NADPH it produces two molecules of glutamate that can yield, via GDH, two NADPH that can in turn serve as an electron source for the anti-oxidative enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR see p. 264-266) glutamine could also be used in pyrimidine biosynthesis. (The glutamate metabolic pathway can be found online at http //sites.huji.ac. il/malaria/maps/glutamatemetpath.html last accessed 16 July 2008.)... 

Aoki, T. and Oya, H. (1979) Glutamine-dependent carbamoyl-phosphate synthetase and control of pyrimidine biosynthesis in the parasitic helminth Schistosoma mansoni. Comp. Biochem. Physiol. 63-B 511-515. 

In the first step of the urea cycle, NH4, bicarbonate, and ATP react to form carbamoyl phosphate (see Fig. 38.12). The cleavage of 2 ATPs is required to form the high-energy phosphate bond of carbamoyl phosphate. Carbamoyl phosphate synthetase I (CPSI), the enzyme that catalyzes this first step of the urea cycle, is found mainly in mitochondria of the liver and intestine. The Roman numeral suggests that another carbamoyl phosphate synthetase exists, and indeed, CPSll, located in the cytosol, produces carbamoyl phosphate for pyrimidine biosynthesis, using nitrogen from glutamine (see Chapter 41). 

The overall scheme of pyrimidine nucleotide biosynthesis differs from that of purine nucleotides in that the pyrimidine ring is assembled before it is attached to ribose-5-phosphate. The carbon and nitrogen atoms of the pyrimidine ring come from carbamoyl phosphate and aspartate. The production of carbamoyl phosphate for pyrimidine biosynthesis takes place in the cytosol, and the nitrogen donor is glutamine. (We already saw a reaction for the production of carbamoyl phosphate when we discussed the urea cycle in Section 23.6. That reaction differs from this one because it takes place in mitochondria and the nitrogen donor is NH/). 

Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD and an ammonia-dependent enzyme (CPSase /) which is localized in the mitochondrial matrix and which is used in urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytoplasm. 

Ito, K. and Uchino, H. Control of pyrimidine biosynthesis in human lymphocytes. Induction of glutamine-utilizing car-bamyl phosphate synthetase and operation of orotic acid pathway during blastogenesis. J. Biol. Chem., 246, 4060-4065 (1971)... 

The cytoplasmic enzyme is active in pyrimidine biosynthesis. Free ammonium ions can replace the glutamine, but only at higher than physiological concentrations. 

Table 5-III lists the glutamine amide transfer reactions of purine and pyrimidine biosynthesis de novo, and of purine and pyrimidine ribonucleotide interconversion, and several more that occur in other areas of metabolism. All have features in common 1, 3).

Acivicin is a potent inhibitor of several steps in purine nucleotide biosynthesis that utilize glutamine. The enzymes it inhibits are glutamine PRPP amidotransferase (step 1, fig. 23.10), phosphoribosyl-A-formylglycinamidine synthase (step 4, fig. 23.10), and GMP synthase (see fig. 23.11). In pyrimidine nucleotide biosynthesis the enzymes inhibited are carbamoyl synthase (step 1, fig. 23.13) and CTP synthase (see fig. 23.14). Acivicin is under trial for the treatment of some forms of cancer. 

The atoms of the pyrimidine ring are derived from carbamoyl phosphate and aspartate, as shown in Fig. 15-14. The de novo biosynthesis of pyrimidine nucleotides is shown in Fig. 15-15. The first completely formed pyrimidine ring is that of dihydroorotate. Only after oxidation to orotate is the ribose attached to produce orotidylate. The compound 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) provides the ribose phosphate. L-Glutamine is used as a substrate donating nitrogen atoms at reactions 1 and 9, catalyzed by carbamoyl phosphate synthetase II and CTP synthetase, respectively a second... 

Biosynthesis of purine and pyrimidine nucleotides requires carbon dioxide and the amide nitrogen of glutamine. Both use an amino acid... 

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. 

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