Carbamoyl phosphate synthetase pyrimidine biosynthesis

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). 

Discuss the structure and mechanism of carbamoyl phosphate synthetase (CPS). Explain the role of carbamoyl phosphate in pyrimidine biosynthesis. 

The liver also receives some ammonia via the portal vein from the intestine, from the bacterial oxidation of amino acids. Whatever its source, the Nib generated in liver mitochondria is immediately used, together with C02 (as HCO3) produced by mitochondrial respiration, to form carbamoyl phosphate in the matrix (Fig. 18-1 la see also Fig. 18-10). This ATP-dependent reaction is catalyzed by carbamoyl phosphate synthetase I, a regulatory enzyme (see below). The mitochondrial form of the enzyme is distinct from the cytosolic (II) form, which has a separate function in pyrimidine biosynthesis (Chapter 22). 

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. 

Unlike in purine biosynthesis, the pyrimidine ring is synthesized before it is conjugated to PRPP. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm, in contrast to the carbamoyl phosphate used in urea synthesis, which is made in the mitochondrion. The enzyme that carries out the reaction is aspartate transcarbamoylase, an enzyme that is closely regulated. 

Carbamoyl phosphate synthetase is part of a multifunctional enzyme that catalyzes the first three steps in pyrimidine biosynthesis. 

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... 

There are two multifunctional proteins in the pathway for de novo biosynthesis of pyrimidine nucleotides. A trifunctional protein, called dihydroorotate synthetase (or CAD, where the letters are the initials of the three enzymatic activities), catalyzes reactions 1, 2 and 3 of the pathway (HCC>5"- CAP— CA-asp—> DHO Fig. 15-15). The enzymatic activities of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase, are contained in discrete globular domains of a single polypeptide chain of 243 kDa, where they are covalently connected by segments of polypeptide chain whch are susceptible to digestion by proteases such as trypsin. A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate— OMP—> UMP Fig. 15-15). Two enzymatic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa which associates as a dimer. 

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. 

We have previously encountered carbamoyl phosphate as a sub- strate for aspartate transcarbamoylase, the enzyme that catalyzes the first step in pyrimidine biosynthesis (Section 10.1). Carbamoyl phosphate synthetase... 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS) (Section 23.4.1). Analysis of the structure of CPS reveals two homologous domains, each of which catalyzes an ATP-dependent step (Figure 25.3). 

Nucleotide biosynthesis is regulated by feedback inhibition in a manner similar to the regulation of amino acid biosynthesis (Section 24,3). Indeed, aspartate transcarbamoylase, one of the key enzymes for the regulation of pyrimidine biosynthesis in bacteria, was described in detail in Chapter 10. Recall ihaiATCase is inhibited by CTP, the final product ofpyrimidine biosynthesis, and stimulated by ATP. Carbamoyl phosphate synthetase is a site of feedback inhibition in both prokaryotes and eukaryotes. 

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. 

UDP-pyridoxal 93 has been prepared in good yield from uridine-5 -phosphoro-morpholidate and pyridoxal phosphate and found to activate the enzyme carbamoyl phosphate synthetase (CPasell) which is involved in pyrimidine biosynthesis. ... 

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 answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

In mammals, carbamoyl phosphate synthetase II is the key regulatory enzyme in the biosynthesis of pyrimidine nucleotides. The enzyme is inhibited by UTP, the product of the pathway, and stimulated by purine nucleotides. In many bacteria, aspartate carbamoyl transferase is the key regulatory enzyme. It is inhibited by CTP and stimulated by ATP. 

UDP-glucose is also an inhibitor of carbamoyl phosphate synthetase II, the primary regulatory enzyme in pyrimidine biosynthesis. 

Carbamoyl phosphate synthetase is an enzyme in pyrimidine biosynthesis that catalyzes the reaction below. 

Carbamoyl phosphate synthetase is the primary regulatory enzyme in pyrimidine biosynthesis of eukaryotes (Figure 11.35). In mammals, carbamoyl phosphate synthetase II is inhibited by the following pyrimidine-containing compounds UDP, UTP, CTP, dUDP, and UDP-glucose. 

In the nematodes the five soluble enzymes of pyrimidine de novo biosynthesis were found in crude extracts of Nippostrongylus brasiliensis and Trichuris muris (81). The first enzyme of de novo synthesis, glutamine-dependent carbamoyl-phosphate synthetase, was deteeted in Ascaris ovary extracts (115), and aspartate transcarbamoylase activity, the second enzyme of de novo synthesis, was found in A. suum (113). 

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. 

Feedback inhibition in pyrimidine nucleotide biosynthesis takes place in several ways. GTP is an inhibitor of aspartate transcarbamoylase and of GTP synthetase. UMP is an inhibitor of an even earlier step, the one catalyzed by carbamoyl phosphate synthetase (Figure 23.29). 

Which of the following statements about the carbamoyl phosphate synthetase of mammals, which is used for pyrimidine biosynthesis, are true ... 

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. 

Birds appear unable to synthesise any arginine via the urea cycle (see Section 5.4), which may be because of lack of carbamoyl phosphate synthetase I in mitochondria (Baker, 1991), and, as a result, the dietary requirement for arginine is higher than in growing mammals. However, they do appear to have a carbamoyl phosphate synthetase II in the cytosol (Maresh, Kwan Kalman, 1969). This may be part of the multienzyme protein G D (carbamoyl phosphate synthase-aspartate carbamoyl transferase-dihydroorotase), responsible for the biosynthesis of 3ihydroorotate, a pyrimidine precursor, but this is bound on the multienzyme protein, and it seems unlikely that it would be available for arginine biosynthesis (see Price Stevens, 1989). 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

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