Carbamyl phosphate, synthesis

Davis, R.H. Carbamyl phosphate synthesis in Neurospora crassa. I. Preliminary characterization of arginine-specific carbamyl phosphokinase. Biochim. Biophys. Acta, 107, 44-53 (1965)... 

In addition to the requirement for pyrimidine nucleotide synthesis, carbamyl phosphate is required for synthesis of arginine and urea. Carbamyl phosphate synthesis is a prominent activity in ureotelic liver and is aimed primarily at the formation of urea the process of urea synthesis is served by a special carbamyl phosphate synthetase which is quite distinct from the enzymes responsible for carbamyl phosphate synthesis in extrahepatic tissues and in the livers of uricotelic animals. A third mechanism for synthesis of carbamyl phosphate is found in bacteria. 

Urinary orotic acid generally is very elevated in babies with OTC deficiency and normal or even low in the infant with CPS deficiency. Patients with OTC deficiency have orotic aciduria because carbamyl phosphate spills into the cytoplasm, where it enters the pathway of pyrimidine synthesis. 

The underlying biochemical defect is a failure of mitochondrial uptake of ornithine. This results in a failure of citrulline synthesis and a consequent hyperammonemia. Urinary orotic acid is high, presumably because of underutilization of carbamyl phosphate. In contrast, excretion of creatine is low, reflecting the inhibition of glycine trans-amidinase by excessive levels of ornithine. 

Carbamyl phosphate synthetase catalyzes the synthesis of carbamyl-P from HCO3-, glutamine, and 2 moles of ATP. The enzyme also catalyzes the HC03 -dependent hydrolysis of ATP. Raushel and Villafranca (5) followed the exchange of from the bridge to the nonbridge position of [y- 0]ATP after Incubation with enzyme and bicarbonate. The exchange rate was O.A times the rate of ADP formation. These results support the formation of carboxy phosphate as the first Intermediate In the catalytic sequence. 

Animal and bacterial enzymes that utilize or synthesize carbamyl phosphate have activity with acetyl phosphate. Acyl phosphatase hydrolyzes both substrates, and maybe involved in the specific dynamic action of proteins. Ornithine and aspartic transcarbamylases also synthesize acetylornithine and acetyl aspartate. Finally, bacterial carbamate kinase and animal carbamyl phosphate synthetase utilize acetyl phosphate as well as carbamyl phosphate in the synthesis of adenosine triphosphate. The synthesis of acetyl phosphate and of formyl phosphate by carbamyl phosphate synthetases is described. The mechanism of carbon dioxide activation by animal carbamyl phosphate synthetase is reviewed on the basis of the findings concerning acetate and formate activation. 

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. 

Propionyl CoA inhibits A(-acetylglutamate synthetase competitively with respect to acetyl CoA, forming A(-propionylglutamate and reducing the synthesis of A(-acetylglutamate. This is an obligatory activator of carbamyl phosphate synthetase, the first enzyme of urea synthesis. Vitamin B12 deficiency may result in some degree of protein intolerance and hyperammonemia. 

The role of ATP in the carboxylation of biotin is unclear. It is possible that biotin is O-phosphorylated during the carboxylation reaction. However, evidence suggests that the immediate reactive species that carboxylates biotin is carboxyphosphate, as in the (biotin-independent) reaction of carbamyl phosphate synthetase in urea and pyrimidine synthesis. 

Citrulline is an endogenous amino acid involved in the urea cycle. Clinically, it can be used as an arginine substitute in the treatment of inborn errors of urea synthesis, including carbamyl phosphate synthetase and ornithine transcarbamylase. It is also a diuretic. 

The first step in the formation of urea from ammonia is its combination with bicarbonate to form carbamyl phosphate (Fig. 1). This contributes only one nitrogen atom to urea, the other being donated by aspartic acid in the third step of the pathway. A -Acetylglutamate is required as cofactor, and the presence of Mg is essential, ATP being converted to ADP in the process. The reaction is catalyzed by carbamyl phosphate synthetase (carbamate kinase EC 2.7.2.2). It has been shown that there are probably two forms of this enzyme, at least in rat liver. One is ammonia dependent, is primarily associated with mitochondria, and may be the enzyme responsible for the formation of carbamyl phosphate in the synthesis of urea. The other, which is glutamine dependent, is probably mainly extramitochondrial and may supply the carbamyl phosphate used... 

The utilization of ammonia resulting from the combination of carbamyl phosphate with aspartic acid, the initial reaction for the synthesis of the pyrimidine nucleotides, continues only as long as there is a requirement for them (Fig. 3). Regulation of this biosynthetic pathway is probably by way of feedback inhibition of aspartate transcarbamylase. The rat liver enzyme is inhibited by uridine, cytidine or thymidine or such derivatives as CMP, UTP, or TMP, all intermediates or products of this pathway (B8). This is not the only enzyme of the pathway which may be subject to feedback regulation. Dihydroorotase from rat liver is also inhibited by some pyrimidines and purines (B9). 

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