Carbamyl phosphate synthetase reaction

McKinley S, Anderson CD, Jones ME. 1967. Studies on the action of hydrazine, hydroxylamine, and other amines in the carbamyl phosphate synthetase reaction. J Biol Chem 14 3381-3390. 

In the cases studied, the ammonia or glutamine reacts with an activated, enzyme-bound acceptor. Thus Meister (4) has suggested that the glutamine-dependent carbamyl phosphate synthetase reaction may follow... 

The positional Isotope exchange has also been measured with 31p-nmr In the reverse reaction of carbamyl phosphate synthetase ... 

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. 

Defects of the enzymes mediating all four reactions of the urea cycle proper have now been established, and there is some evidence of the existence of a fifth enzyme defect, involving carbamyl phosphate synthetase, mediating the initial reaction of the pathway. As the first report of a metabolic disorder involving the urea cycle was only in 1958, it is not surprising that there have been very few reviews of this topic, that of Efron (El) being the most complete to date. 

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 substances assayed in the reaction mixture are citrulline, in determining carbamyl phosphate synthetase and ornithine transcarbamylase, and urea in determining argininosuccinate synthetase, argininosuccinate lyase, and arginase. 

The st example concerns the intracellulEir localization of the reactions of the ornithine cycle. The enzymes catalyzing two of the reactions, carbamyl phosphate synthetase and ornithine transcarbamylase, Eire found in the mitochondrial matrix while the other three enzymes are in the cjrtosol. As a result, the mitochondrial membrane is interposed as a barrier to the operation of the cycle, necessitating two addi-... 

This reaction is irreversible and requires two molecules of ATP Af-acetyl-glutamate is required in this reaction as an allosteric cofactor. Glutamine will not replace ammonia in this reaction. The enzyme will be referred to as carbamyl phosphate synthetase I. 

As in other reactions in which either ammonia or the amide group of glutamine is utilized (amination of UTP to form CTP, synthesis of NAD from desamido-NAD, and of guanylate from xanthylate), the carbamyl phosphate synthetase II reaction is inhibited by the glutamine analogues, azaserine and diazo-oxo-norleucine (see Chapter 5). 

The cycle starts with carbamyl phosphate formation (this reaction was discussed in the section on pyrimidine biosynthesis). Carbamyl phosphate synthetase catalyzes the condensation of active CO2 with NH4 to yield carbamyl phosphate, a precursor of pyrimidines and urea. 

The substrate structures and chemical reactions participating in the conversion of citrulline to arginine are given in Fig. 3. The intermediate formed in the conversion of ornithine to citrulline was found by Grisolia and Cohen in 1952. The structure of this compound was shown by chemical synthesis to be carbamylphosphate by Jones, Spector and Lipmann in 1955. Subsequent advances on carbamyl phosphate synthetase and ornithine transcarbamylase in the laboratories of P.P. Cohen, S. Grisolia, P. Reichard, M. E. Jones and M. Marshall have contributed to the enzymology and elucidation of the reaction mechanisms (cf. Fig. 3). The two enzymes are localized within the liver mitochondria, the last three enzymes of the cycle are in the cytosol. 

Liver of true ureotelic animals (see 401a) contmns the enzyme carbamyl phosphate synthetase which catalyzes the following reaction (2, 402) ... 

The enzymes discussed in the previous sections (carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, cleavage enzyme, and arginase) constitute the known enzymic steps in the sequence of reactions leading to the biosynthesis of urea in ureotelic animals in accordance with the cycle originally proposed by Krebs and Hen-seleit (458). A summary scheme showing the steps in this cycle and the relationship of some of the intermediates to other systems is shown in Fig. 2. 

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. 

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). 

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