Ammonia, carbamoyl phosphate from

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS) (Section 23.4.1). Analysis of the structure of CPS reveals two homologous domains, each of which catalyzes an ATP-dependent step (Figure 25.3). 

Urea is synthesized in the liver in the urea cycle. The first step is formation of carbamoyl phosphate from ammonia, C02, and ATP. This is followed by a number of other steps, including formation of citrulline, argininosuccinate, and arginine, which is split to urea plus ornithine. The second nitrogen of urea is donated by aspartate in the formation of argininosuccinate. 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

CPS.A and CPS.B are each comprised of three smaller subdomains (Figure 2), Al, A2, A3 and Bl, B2, B3, respectively. Separately cloned A2 and B2 are catalytic subdomains (20) that can catalyze the formation of carbamoyl phosphate from NH3, ATP and bicarbonate. While these species dimerize, they lack intermolecular tunnels and have a catalytic mechanism similar to carbamate kinases that synthesize carbamoyl phosphate by the phosphorylation of carbamate formed chemically from ammonia and bicarbonate in solution. The designation of A2 and B2 as catalytic subdomains is consistent with the x-ray structure of the E. coli enzyme that showed ADP and an ATP analogue bound to these bilobal subdomains (26,70). The function of A3 is unknown, while B3, as discussed below, is the major locus of regulation. Comparison of the kinetics of A1-A2 and A2 suggest that Al is an attenuation subdomain that suppresses the catalytic activity of A2. As in the case of the GLN domain, the coordination of reactions occurring on the GLN, CPS.A and CPS.B requires a mechanism that... 

FIGURE 20.4 Mechanism of the formation of carbamoyl phosphate from bicarbonate. Bicarbonate ion is first activated by phosphorylation with ATP, and a nucleophilic acyl substitution with ammonia then occurs. 

From ammonia through carbamoyl phosphate synthetase To urea... 

The carbamoyl phosphate synthetase (abbreviated to CPS-I) that is involved in the ornithine cycle differs from the enzyme that is involved in pyrimidine synthesis (carbamoyl phosphate synthetase-ll). The latter enzyme is cytosolic, requires glutamine for provision of nitrogen, rather than ammonia, and is regulated by different factors (Chapter 20). 

The liver also receives some ammonia via the portal vein from the intestine, from the bacterial oxidation of amino acids. Whatever its source, the Nib generated in liver mitochondria is immediately used, together with C02 (as HCO3) produced by mitochondrial respiration, to form carbamoyl phosphate in the matrix (Fig. 18-1 la see also Fig. 18-10). This ATP-dependent reaction is catalyzed by carbamoyl phosphate synthetase I, a regulatory enzyme (see below). The mitochondrial form of the enzyme is distinct from the cytosolic (II) form, which has a separate function in pyrimidine biosynthesis (Chapter 22). 

MECHANISM FIGURE 18-11 Nitrogen-acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (a) in the reaction catalyzed by carbamoyl phosphate synthetase 1, the first nitrogen enters from ammonia. The terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. In other words, this reaction has two activa-... 

Ammonia is highly toxic to animal tissues. In the urea cycle, ornithine combines with ammonia, in the form of carbamoyl phosphate, to form citrulline. A second amino group is transferred to citrulline from aspartate to form arginine—the immediate precursor of urea. Arginase catalyzes hydrolysis of arginine to urea and ornithine thus ornithine is regenerated in each turn of the cycle. 

One of the nitrogens enters the urea cycle via carbamoyl phosphate that comes from ammonia. The second urea nitrogen enters the urea cycle as part of aspartic acid that can come from transamination of oxaloacetate. Glutamate is the source of the amino group in the transamination. 

Carbamoyl phosphate synthetase, which is technically not a member of the urea cycle, catalyzes the condensation and activation of ammonia (from the oxidative deamination of glutamate by glutamate dehydrogenase Topic M2) and C02 (in the form of bicarbonate, HC03 ) to form carbamoyl phosphate. The hydrolysis of two ATP molecules makes this reaction essentially irreversible. 

Like the synthesis of carbamoyl phosphate, this reaction requires ATP and uses glutamine as the source of the amino group. The reaction proceeds through an analogous mechanism in which the 0-4 atom is phosphorylated to form a reactive intermediate, and then the phosphate is displaced by ammonia, freed from glutamine by hydrolysis. CTP can then be used in many biochemical processes, including RNA synthesis. 

Carbamoyl Phosphate Synthetase 11 (CPS-11) differs in several ways from its isoform (CPS-I), the enzyme which provides carbamoyl phosphate for the Urea cycle (see "Protein Turnover / Ammonia Metabolism"). 

Figure 25.3 Structure of carbamoyl phosphate synthetase. Notice that the enzyme contains sites for three reactions. This enzyme consists of two chains. The smaller chain (yellow] contains a site for glutamine hydrolysis to generate ammonia. The larger chain includes two ATP grasp domains (blue and red). In one ATP-grasp domain (blue), bicarbonate is phosphorylated to carboxyphosphate. which then reacts with ammonia to generate carbarrric acid. In ihe other ATP-grasp domain, the carbamic acid is phosphorylated to produce carbamoyl phosphate. [Drawn from lJDB.pdb.]...

Figure 11 The structure of carbamoyl phosphate synthase, showing in gray mesh the tunnel that transfers ammonia released from glutamine by the small subunit (blue) to an active site in the N-terminal domain of the large subunit (green), where it reacts with carbonate and ATP to form carbamate. The carbamate then travels through the tunnel to a third active site in the C-terminal domain of the large subunit (purple), where it is phosphorylated by ATP to form carbamoyl phosphate. Reproduced with permission from A. Weeks L. Lund F. M. Raushel, Cuir. Opin. Chem. Biol. 2006, 10, 465-472.

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