Carbamoyl phosphate synthetase active sites

For dihydroorotate synthetase, the product of reaction 1, carbamoyl phosphate (CAP) is very unstable but is rapidly transformed by aspartate transcarbamoylase which is 50 times more active (per active site) than carbamoyl phosphate synthetase. High levels of carbamoyl aspartate (CA-asp) may be toxic, but this intermediate is rapidly consumed by the high dihydroorotase activity. Because the first three reactions are catalyzed by a single protein, the three enzyme active sites are expressed in a constant ratio under all conditions of growth this maintains CAP and CA-asp at low levels. For UMP synthase, OMP decarboxylase is far more active (per active site) than orotate PRTase, resulting in low cellular levels of the intermediate, OMP, which would otherwise be subject to enzymatic hydrolysis (in cells from higher animals). 

The active site for this reaction lies in a domain formed by the aminoterminal third of CPS. This domain forms a structure, called an ATP-grasp fold, that surrounds ATP and holds it in an orientation suitable for nucleophilic attack at the Y phosphoryl group. Proteins containing ATP-grasp folds catalyze the formation of carbon-nitrogen bonds through acyl-phosphate intermediates and are widely used in nucleotide biosynthesis. In the final step catalyzed by carbamoyl phosphate synthetase, carbamic acid is phosphorylated by another molecule of ATP to form carbamoyl phosphate. 

Figure 25.4. Ammonia-Generation Site. The smaller domain of carbamoyl phosphate synthetase contains an active site...

Figure 25.5. Substrate Chanueliug. The three active sites of carbamoyl phosphate synthetase are linked by a channel (yellow) through which intermediates pass. Glutamine enters one active site, and carbamoyl phosphate, which includes the nitrogen atom from the glutamine side chain, leaves another 80 A away.

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 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. 

Carbamoyl phosphate synthetase contains three different active sites (see... 

The regulated step of pyrimidine synthesis in humans is carbamoyl phosphate synthetase 11. The enzyme is inhibited by UTP and activated by PRPP (see Fig. 41.14). Thus, as pyrimidines decrease in concentration (as indicated by UTP levels), CPS-11 is activated and pyrimidines are synthesized. The activity is also regulated by the cell cycle. As cells approach S-phase, CPS-11 becomes more sensitive to PRPP activation and less sensitive to UTP inhibition. At the end of S-phase, the inhibition by UTP is more pronounced, and the activation by PRPP is reduced. These changes in the allosteric properties of CPS-11 are related to its phosphorylation state. Phosphorylation of the enzyme at a specific site by a MAP kinase leads to a more easily activated enzyme. Phosphorylation at a second site by the cAMP-dependent protein kinase leads to a more easily inhibited enzyme. 

Although aspartate carbamoyltransferase appears to be a control site in the synthesis of pyrimidines in E, coli (and probably other microorganisms) the corresponding transferase in animal cells does not appear to be a regulatory enzyme. Furthermore, since its activity in various tissues is far greater than that of carbamoyl phosphate synthetase II, animal aspartate carbamoyltransferase is not the rate-limiting enzyme in the pathway. 

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