Carbamyl phosphate and

Biosynthesis and Utilization of Acetyl Phosphate, Formyl Phosphate, and Carbamyl Phosphate and their Relations to the Urea Cycle... 

Stadtman has demonstrated (42) the conversion of carbamyl phosphate and acetate to acetyl phosphate, catalyzed either by a crude lysine system or by a purified acetate kinase from C. sticklandii. ADP and... 

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. 

The condensation of carbamyl phosphate and L-aspartate, catalyzed by aspartate trans-carbamoylase (ATCase), produces iV-carba-myl-L-aspartate (Equation 17.38). This is one of the early steps in de novo pyrimidine biosynthesis, also a requirement for cell division. 

In this method, a blank containing an inhibitor is necessary since carbamyl phosphate will transfer its carbamyl group not only to ornithine, but also to the glycylglycine used for the buffer, and because there is a slow chemical combination of carbamyl phosphate and ornithine. The error is too small to be detectable by the color reaction of Brown and Cohen, but large enough to be apparent when the more sensitive reagent is used. The blank contains all the reactants, with the addition of phenyl mercuric borate (Famosept), which inhibits the enzyme-catalyzed formation of citrulline, but has no effect on its noncatalyzed chemical formation. 

Levin et al. has shown that whereas the ornithine transcarbamylase deficiency is severe and critical in most cases of hyperammonemia, in one child, a boy, the enzyme activity was reduced to a lesser extent (L6). Further investigations showed that other differences between the enzyme in this child and in others affected, existed. Thus the affinities of the enzyme for both carbamyl phosphate and ornithine were markedly different in this child from the others. On the basis of these findings they suggested that this constituted a distinct genetic variant, which could explain its occurrence in a male infant whereas all other reported cases of hyperammonemia were in females. 

Figure 7. Interactions at the active site of aspartate transcarbamylase (ATCase). N-phosphonoacetyl-L-asparate (PALA) is a bisubstrate analog of the two natural substrates of ATCase, carbamyl phosphate and L-aspartate. PALA is shown bound in the active site of ATCase. Noncovalent interactions between PALA and side-chains of the protein are shown as dashed lines. Specific residues are indicated by their one letter abbreviation and by their position in the protein sequence (e.g., HI 34 = histidine at position 134). The active site is composed of residues from two separate polypeptide chains (denoted by primed and unprimed residue numbers). Note the complimentarity of the site and the ligand. The same interactions are used to align and catalyze the condensation of ATCase s natural substrates (Monaco et al., 1978).

Pyrimidine biosynthesis commences with a reaction between carbamyl phosphate and aspartic acid to give carbamyl aspartic acid which then nndergoes ring closure and oxidation to orotic acid. A reaction then occurs between orotic acid and 5-phosphoribosyl pyrophosphate to give orotidine-5-phosphate which on decarboxylation yields uridine-5-phosphate (UMP). By means of two successive reactions with ATP, UMP can then be converted into UTP and this by reaction with ammonia can give rise to cytidine triphosphate, CTP (11.126). 

Another form of spatial organization of metabolism that is often seen in eukaryotes but is less common in bacteria involves enzyme aggregates or multifunctional enzymes. An example is seen in S. cerevisiae where the first two reactions in pyrimidine nucleotide biosynthesis, the synthesis of carbamyl phosphate and the carbamylation of aspartate, are catalyzed by a single bifunctional protein (31). Both reactions are subject to feedback inhibition by UTP, in contrast to the situation inB. subtilis where aspartate transcarbamylase activity is not controlled. It is possible that an evolutionary advantage of the fusion of the genes... 

Reichard found that carbamyl aspartate synthesis would take place in crude liver preparations if carbamyl phosphate and aspartate were provided and showed that the enzyme responsible, aspartate carbamyltransferase, was not the same as that which formed citrulline, but apparently shared carbamyl phosphate with it. 

L-Arginine originates from L-ornithine, carbamyl phosphate and the amino group of L-aspartic acid (Fig. 236). 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

While the studies of Boyland and Roller and Elion and co-workers, which were conducted in vivo, do suggest that urethane has a specificity for pyrimidine biosynthesis, Kaye could not demonstrate in vitro any significant inhibition by urethane of several enzymes involved in nucleic acid metabolism. Both urethane and its A -hydroxy metabolite bear a structural resemblance to carbamyl phosphate and carbamyl-L-aspartate. The enzyme aspartate transcarbamylase begins pyrimidine biosynthesis by catalyzing the formation of carbamyl-L-aspartate from carbamyl phosphate and l-aspartate. Giri and Bhide have reported that in vivo administration of urethane decreased aspartate transcarbamylase activity of lung tissue of adult male and (to a lesser extent) female mice no in vitro inhibition could be demonstrated. 

In preceding sections of this chapter, the important metabolic reactions which yield ammonia have been discussed. Certain of these systems are capable of fixing ammonia (glutamic dehydrogenase, alanine dehydrogenase, L-amino acid oxidase, etc.). The fixation of ammonia in the glutamine synthetase system will be discussed in Chapter 17. The present section will deal with (a) enzymes which fix ammonia to form carbamyl phosphate and (b) enzymes which utilize carbamyl phosphate for the synthesis of arginine (and urea) and pyrimidines. 

Because carbamate is considered to be involved, the term carbamate kinase may be used for this enzyme. The reaction is freely reversible which indicates that the free energy of hydrolysis of carbamyl phosphate and ATP are of the same order of magnitude. 

The formation of carbamylaspartic acid from carbamyl phosphate and aspartic acid (Fig. 20) has been demonstrated in pigeon liver, several tissues of the rat, E. colt, and yeast (363S67). It appears that only one enz3rme was involved in thb interesting reaction 368) and it has been named ureidosuccinic (carbamylaspartic) acid synthetase 355). The reaction was essentially irreversible, even though slight reversal was shown with labeled substrates 357). 

The splitting of dihydrouracil to j8-ureidopropionic acid was reversible and occurred in the livers of the calf, rat, and pigeon, but was not detected in brain, muscle, and heart of the rat, or baker s and brewer s yeast (437). The further conversion of jS-ureidopropionic acid to /8-alanine, carbon dioxide, and ammonia was not reverrible by a single enzymic mechanism. However, upon addition of carbamyl phosphate and /3-alanine to varous systems, the syntheris of /9-ureidopropionic acid was observed (430, 437). Both dihydrouracil and /3-ureidopropionic acid were utilized for RNA... 

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