Formation of Carbamoyl Phosphate

Carbamoyl phosphate synthesis requires amino acid acetyltransferase (N-acetylglutamate synthase, mitochondrial) and carbamoyl-phosphate synthase I (CPSI). N-Acetylglutamate (NAG) is an obligatory positive effector of CPSI. NAG synthase is under positive allosteric modulation by arginine and product inhibition by NAG. Depletion of CoA-SH decreases NAG synthesis and ureage-nesis. This situation can occur in organic acidemias (e.g., propionic acidemia Chapter 18), in which organic acids produced in excess compete for CoA-SH for formation 

NAG binding changes the conformation and subunit structure of CPSI, with preponderance of the monomers. Carbamoylglutamate is also an activator of CPSI. Glutamate and a-ketoglutarate compete with NAG for binding. CPSI is subject to product inhibition by Mg-ADP. It 

In humans, there are two immunologically distinct carbamoyl phosphate synthases, one mitochondrial (CPSI) and the other cytosolic (CPSII). CPSI is involved in ure-agenesis, uses NH3 exclusively as the nitrogen donor, and requires binding of NAG for activity. CPSII uses glutamine as substrate, is not dependent on NAG for activity, and is required for synthesis of pyrimidine 

The condensation of citrulline and aspartate to argininosuccinate is catalyzed by argininosuccinate synthase in the cytosol and occurs in two steps. In the initial step, the ureido group is activated by ATP to form the enzyme-bound intermediate adenylylcitrulline. In the second step, nucleophilic attack of the amino group of aspartate displaces AMP and yields argininosuccinate. The overall reaction is shown below  

The reaction is driven forward by hydrolysis of pyrophosphate to inorganic phosphate. Argininosuccinate formation is considered as the rate-limiting step for urea synthesis. This reaction incorporates the second nitrogen atom of the urea molecule donated by aspartate. 

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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... 

The activity of the urea cycle is regulated at the level of enzyme synthesis and by allosteric regulation of the enzyme that catalyzes the formation of carbamoyl phosphate. 

Carbamoyl phosphate synthetases. The first of the individual steps in the urea cycle is the formation of carbamoyl phosphate.163 Carbon dioxide and ammonia equilibrate spontaneously with carbamic acid ... 

The mechanism of formation of carbamoyl phosphate. The reaction involves three steps, all of which take place on the same enzyme, carbamoyl phosphate synthase. 

Xanthine does not react with PRPP. ATP reacts in the formation of carbamoyl phosphate, orotic acid in the pyrimidine pathway, and uracil and hypoxanthine in the salvage reactions. 

CPSI catalyzes the formation of carbamoyl phosphate from bicarbonate, ammonium, and two adenosine triphosphate molecules (Fig. 18-1).This first step of the urea cycle occurs in the mitochondrial matrix and assimilates the first of the two nitrogen atoms that will eventually be found in urea. While two ATP molecules are hydrolyzed, there is formation of a lower energy bond in carbamoyl phosphate. CPSI is a homodimer that accounts for 15-30% of the total protein mass in liver mitochondria. jV-Acetylglutamate (NAG) is an essential allosteric activator of CPSI activity, and magnesium ions are also required for its activity. 

Urea is synthesized in the liver by a series of reactions known as the urea cycle (Fig. 15-13). One nitrogen is derived from ammonium, the second from aspartate and the carbon is derived from C02. The synthesis of urea requires the formation of carbamoyl phosphate and the four enzymatic reactions of the urea cycle. Some of the reactions take place in the mitochondria and some in the cytoplasm. The enzymes involved in the synthesis of urea are discussed below. 

The formation of carbamoyl phosphate (NH2COOPO3-) takes place in the matrix of mitochondria ... 

The Urea Cyele Begins with the Formation of Carbamoyl Phosphate... 

Biotin is a coenzyme for the carbon dioxide fixation reactions catalyzed by acetyl-CoA carboxylase (Chapter 19), propionyl-CoA carboxylase, pyruvate carboxylase, and S-methylcrotonyl-CoA carboxylase. Car-boxylation reactions that do not require biotin are the addition of Ce to the purine ring (Chapter 27), the formation of carbamoyl phosphate (Chapter 17), and the y-carboxylation of glutamyl residues of several of the clotting factors, which requires vitamin K (Chapter 36). 

The answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

Urea synthesis, which occurs in hepatocytes, begins with the formation of carbamoyl phosphate in the matrix of mitochondria. The substrates for this reaction, catalyzed by carbamoyl phosphate synthetase I, are NH4 and HCO3. (The nitrogen source for carbamoyl phosphate synthetase II, the enzyme involved in pyrimidine synthesis, is glutamine.)... 

The first two reactions in the biochemical pathway that converts NFL,+ to urea (i.e., the formation of carbamoyl phosphate and citrulline) occur in the mitochondrial matrix. Subsequent reactions that convert citrulline to ornithine and urea occur in the cytosol. Both citrulline and ornithine are transported across the inner membrane by specific carriers. 

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. 

Pyrimidines synthesis occurs in almost all cells. The first step ofpyrimidine synthesis is the formation of carbamoyl phosphate. In the case of pyrimidine synthesis, carbamoyl phosphate is formed using the amide nitrogen of glutamine, C02, and two ATPs (the use of two ATPs assures that the reaction is driven in a forward direction). 

Carbamoyl phosphate formation - Carbamoyl Phosphate Synthetase catalyzes the formation of carbamoyl phosphate in the following two reactions (glutamine is the preferred substrate) ... 

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... 

The reaction is catalysed by carbamoyl phosphate syndiase which is Mg -dependent and has an absolute requirement for N-acetylglutamate as a cofactor. Carbamoyl phosphate synthase is a mitochondrial enzyme and comprises approximately 15% of the matrix protein in liver mitochondria. As a consequence of the hydrolysis of two molecules of ATP, the formation of carbamoyl phosphate is effectively irreversible, so that any ammonia undergoing this reaction is committed to excretion. 

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. 

Figure 16.S illustrates the reactions and the com-partmentalization of the enzymes of the urea cycle. The first reaction in urea biosynthesis is the mitochondrial formation of carbamoyl phosphate, the substrate of the urea cycle. The reaction utilizes an ammonium (NH4 ) ion, delivered into the mitochondrion as glutamate by the action of both the glutamate-aspartate (Section 11.3) and the glutamate-hydroxyl ion antiport carriers. Oxidative deamination of glutamate by glutamate dehydrogenase releases an NH4 ion.

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