Carbamoyl phosphate

Consider carbamoyl phosphate, a precursor in the biosynthesis of pyrimidines ... 

Based on the discussion of high-energy phosphates in this chapter, would you expect carbamoyl phosphate to possess a high free energy of hydrolysis Provide a chemical rationale for your answer. 

One of the steps in the biosynthesis of uridine monophosphate is the reaction of aspartate with carbamoyl phosphate to give carbamoyl aspartate followed by cyclization to form dihydroorotate. Propose mechanisms for both steps. 

Carbamoyl Phosphate Synthase I Initiates Urea Biosynthesis... 

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... 

Carbamoyl Phosphate Plus Ornithine Forms Citrulline... 

L-Ornithine transcarbamoylase catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline and orthophosphate (reaction 2, Figure 29-9). While the reaction occurs in the mitochondrial matrix, both the formation of ornithine and the subsequent metabolism of citmlline take place in the cytosol. Entry of ornithine into mitochondria... 

Carbamoyl Phosphate Synthase I Is the Pacemaker Enzyme of the Urea Cycle... 

The activity of carbamoyl phosphate synthase I is determined by A -acetylglutamate, whose steady-state level is dictated by its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate. These reactions are catalyzed by A -acetylglu-tamate synthase and A -acetylglutamate hydrolase, respectively. Major changes in diet can increase the concentrations of individual urea cycle enzymes 10-fold to 20-fold. Starvation, for example, elevates enzyme levels, presumably to cope with the increased production... 

Hyperammonemia Type 1. A consequence of carbamoyl phosphate synthase I deficiency (reaction 1, Figure 29-9), this relatively infrequent condition (estimated frequency 1 62,000) probably is familial. 

Changes in enzyme levels and allosteric regulation of carbamoyl phosphate synthase by A -acetylglutamate regulate urea biosynthesis. 

Figure 34-7 summarizes the roles of the intermediates and enzymes of pyrimidine nucleotide biosynthesis. The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthase II, a different enzyme from the mitochondrial carbamoyl phosphate synthase I of urea synthesis (Figure 29-9). Compartmentation thus provides two independent pools of carbamoyl phosphate. PRPP, an early participant in purine nucleotide synthesis (Figure 34-2), is a much later participant in pyrimidine biosynthesis.

Five of the first six enzyme activities of pyrimidine biosynthesis reside on multifunctional polypeptides. One such polypeptide catalyzes the first three reactions of Figure 34-2 and ensures efficient channeling of carbamoyl phosphate to pyrimidine biosynthesis. A second bifunctional enzyme catalyzes reactions 5 and 6. 

The orotic aciduria that accompanies Reye s syndrome probably is a consequence of the inabifity of severely damaged mitochondria to utifize carbamoyl phosphate, which then becomes available for cytosofic overproduction of orotic acid. Type I orotic aciduria reflects a deficiency of both orotate phosphoribosyltransferase and orotidylate decarboxylase (reactions 5 and 6, Figure 34—7) the rarer type II orotic aciduria is due to a deficiency only of orotidylate decarboxylase (reaction 6, Figure 34-7). 

Excess carbamoyl phosphate exits to the cytosol, where it stimulates pyrimidine nucleotide biosynthesis. The resulting mild orotic aciduria is increased by high-nitrogen foods. 

Since pyrimidine catabolites are water-soluble, their overproduction does not result in clinical abnormalities. Excretion of pyrimidine precursors can, however, result from a deficiency of ornithine transcar-bamoylase because excess carbamoyl phosphate is available for pyrimidine biosynthesis. 

Arginine deiminase (ADI) pathway (Mercenier et al. 1980) arginine —> citrulline ornithine + carbamoyl phosphate... 

As for the acetyl phosphate monoanion, a metaphosphate mechanism has also been proposed 78) for the carbamoyl phosphate monoanion 119. Once again, an intramolecular proton transfer to the carbonyl group is feasible. The dianion likewise decomposes in a unimolecular reaction but not with spontaneous formation of POf as does the acetyl phosphate dianion, but to HPOj and cyanic acid. Support for this mechanism comes from isotopic labeling proof of C—O bond cleavage and from the formation of carbamoyl azide in the presence of azide ions. 

The search for routes to phosphorylated nucleosides or phosphorylated ribose started, as mentioned in Sect. 4.7.3, with work done by Ponnamperuma (1963). The synthesis of ATP was carried out by phosphorylation of ADP using carbamoyl phosphate in the presence of Ca2+ ions as catalyst yields of up to about 20% were obtained (Saygin and Ellmauerer, 1984). 

Martinez-Ramon, A., Knecht, E., Rubio, V., and Grisolia, S. (1990) Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures. J. Histochem. Cytochem. 38, 371-376. 

From ammonia through carbamoyl phosphate synthetase To urea... 

A. Pi/ erard, Control of the activity of Escherichia coli carbamoyl phosphate synthetase by antagonistic allosteric effectors. Science 154, 1572 1573 (1966). 

Jones, M.E., Spector, L., Lipmann, F. (1955). Carbamoyl Phosphate. In Proceedings of the 3rd International Congress of Biochemistry. (1956) (Liebecq, C., Ed.) Vaillant-Carmanne, Liege. 

Some of the results obtained by differential centrifugation showed enzyme distribution between different cell fractions which were difficult to interpret. Enzymes like carbamoyl phosphate synthase or isocitrate dehydrogenase were found both in mitochondria and in the soluble fraction of the cell. This led to detailed kinetic studies with purified enzymes which indicated there might be populations of enzymes with slightly different properties (isozymes) catalyzing similar reactions in different compartments or in different cell types. Variations in kinetic behavior appeared to tailor the enzyme appropriately to the particular compartment or cell where the reaction took place. 

The ammonia liberated by GLDH does not itself enter the urea cycle it must first be combined with carbon dioxide to form carbamoyl phosphate. This is an energy (ATP) consuming reaction ... 

The enzyme carbamoyl phosphate synthase (CPS) is a control point in the process. Stage 3. The urea cycle (Figure 6.7)... 

Carbonic anhydrase (CA, also called carbonate dehydratase) is an enzyme found in most human tissues. As well as its renal role in regulating pH homeostasis (described below) CA is required in other tissues to generate bicarbonate needed as a co-substrate for carboxylase enzymes, for example pyruvate carboxylase and acetyl-CoA carboxylase, and some synthase enzymes such as carbamoyl phosphate synthases I and II. At least 12 isoenzymes of CA (CA I—XII) have been identified with molecular masses varying between 29 000 and 58 000 some isoenzymes are found free in the cytosol, others are membrane-bound and two are mitochondrial. 

HypF 81.5 Soluble, monomer. Possible acylphosphatase domain. No metal, putative zinc finger Synthesis of CO and CN ligands from carbamoyl phosphate... 

---