Carbamoyl phosphate synthetase II

Carbamoyl-phosphate synthetase (ammonia) [EC 6.3.4.16], also known as carbamoyl-phosphate synthetase I, catalyzes the reaction of two molecules of ATP with carbon dioxide, ammonia, and water to produce two molecules of ADP, orthophosphate, and carbamoyl phosphate. Carbamoyl-phosphate synthetase (glutamine-hydrolyzing) [EC 6.3.5.5], also known as carbamoyl-phosphate synthetase II, catalyzes the reaction of two molecules of ATP with carbon dioxide, glutamine, and water to produce two molecules of ADP, orthophosphate, glutamate, and carbamoyl phosphate. 

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. 

The regulated step of this pathway in mammalian cells is the synthesis of carbamoyl phosphate from glutamine and C02, catalyzed by carbamoyl phosphate synthetase II (CPS U). CPS II is inhibited... 

The reaction is catalyzed by carbamoyl phosphate synthetase II (also called carbamoyl phosphate synthetase, glutamine). 

The reaction of carbamoyl phosphate with aspartate [Figure 10.9, reaction (1)] is rate controlling and committing in microorganisms, whereas in mammals carbamoyl phosphate synthetase II seems to the major rate-controlling enzyme. The latter is stimulated by PRPP and purine nucleotides (especially ATP) and... 

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... 

The end-product of the pathway, UTP, is a potent inhibitor of carbamoyl phosphate synthetase II and the substrate, ATP, also activates this enzyme. The enzymatic activity of carbamoyl phosphate synthetase II is low relative to subsequent enzymes in the pathway (Fig. 15-15) and, under normal conditions, flux through the de novo pathway may be regulated by cellular levels of P-Rib-PP, UTP and ATP i.e., carbamoyl phosphate synthetase II catalyzes the flux-controlling step in the pathway (see Section 10.5). 

Fig. 15-15 The de novo pyrimidine biosynthetic pathway. CAP, carbamoyl phosphate CA-asp, /V-carbamoyl-L-aspartate DHO, L-dihydroorotate Oro, orotate OMP, orotidine 5 -monophosphate. Enzymes (1) carbamoyl phosphate synthetase II (2) aspartate transcarbamoylase (3) dihydroorotase, (4) dihydroorotate dehydrogenase (5) orotate phosphoribosyltransferase (6) OMP decarboxylase (7) nucleoside monophosphate kinase (8) nucleoside diphosphate kinase (9) CTP synthetase.

The primary site of regulation is Carbamoyl Phosphate Synthetase II (glutamine) which is allosterically inhibited by UTP. Elevated PRPP increases the CPS-II activity to help control PRPP levels. Feedback inhibition (control) is provided by TDP inhibition of PRPP synthesis and UMP inhibition of OMP Decarboxylase. 

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. 

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. 

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. 

In mammalian tissue, the six enzymes are encoded by three genes. One gene codes for a multifunctional polypeptide (Pyr 1-3) that is located in the cytosol and has carbamoyl phosphate synthetase II... 

Schematic representation of the intracellular location of the six enzymes of UMP biosynthesis in animals. Pyr 1-3 = 1, Carbamoyl phosphate synthetase II 2, aspartate transcarbamoylase 3, dihydroorotase 4, dihydroorotate dehydrogenase Pyr 5,6 = 5, orotate phosphoribosyltransferase 6, orotidine-5 -monophosphate decarboxylase.

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. 

Urea synthesis, which occurs in hepatocytes, begins with the formation of carbamoyl phosphate in the matrix of mitochondria. The substrates for this reaction, catalyzed by carbamoyl phosphate synthetase I, are NH4 and HCO3. (The nitrogen source for carbamoyl phosphate synthetase II, the enzyme involved in pyrimidine synthesis, is glutamine.)... 

Glutamine + ATP + HC03" <=> Glutamate + Carbamoyl Phosphate + ADP (catalyzed by Carbamoyl Phosphate Synthetase II)... 

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

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. 

See also Urea Cycle Descriptions, Figure 11.35, Carbamoyl Phosphate Synthetase II, Utilization of Ammonia... 

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... 

All of the enzyme activities involved in de novo UMP biosynthesis have been identified in the trematodes Schistosoma mansoni (81,106,107) and S. japonicum (108). Carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydro-orotase have been partially purified from the cytosol and appear to exist, as in mammalian cells, as a multienzyme protein (81,106). Dihydro-orotate oxidase is membrane bound (81), but its electron acceptor has not been identified. Orotate PRTase and OMP decarboxylase are also cytosolic. In mammalian cells these last two enzymes exist as part of a multienzyme protein that efficiently channels orotate to UMP since little free OMP is present in the cell. In S. mansoni they also exist as a multienzyme protein but apparently the channeling is less efiBcient since levels of free OMP are significantly higher than those of UMP (107). 

Fig. 41.14. Synthesis of the pyrimidine bases. CPSn = carbamoyl phosphate synthetase II. RR = ribonucleotide reductase = stimulated by 0 = inhibited by FH2 and FH4 = forms of folate.

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