Pyrimidine Aspartic acid synthetase

The operation of the feedback mechanism in the cell can be described in the following manner if nucleic acid syntheris were proceeding at a rapid rate, the concentration of nucleotides in the cell, e.g. CMP, would decrease, relieve aspartate-carbamyl transferase (also called carbamyl-aspartic acid synthetase) reaction of any inhibition, and thus permit an increased rate of pyrimidine biosyntheris. If nucleic acid syntheris were slowed or halted, the CMP concentration would rise, inhibit the enzyme, and thereby decelerate pyrimidine biosynthesis. 

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

The two conditions can be distinguished by an increase in orotic add and uracil, which occurs in ornithine transcarbamoylase deficiency, but not in the defldency of carbamoyl phosphate synthetase. Orotic acid and uracil are intermediates in pyrimidine synthrais (see Chapter 18). This pathway is stimulated by the accumulation of carbamoyl phosphate, the substrate for ornithine transcarbamoylase in the urea cycle and for aspartate transcarbamoylase in pyrimidine synthesis. 

Eukaryotic organisms contain a multifunctional enzyme with carbamoylphosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activities. Two mechanisms control this enzyme. First, control at the level of enzyme synthesis exists the transcription of the gene for the enzyme is reduced if an excess of pyrimidines is present. Secondly, control exists at the level of feedback inhibition by pyrimidine nucleotides. This enzyme is also an example of the phenomenon of metabolic channeling aspartate, ammonia, and carbon dioxide enter the enzyme and come out as orotic acid. 

Nucleotide biosynthesis is regulated by feedback inhibition in a manner similar to the regulation of amino acid biosynthesis (Section 24,3). Indeed, aspartate transcarbamoylase, one of the key enzymes for the regulation of pyrimidine biosynthesis in bacteria, was described in detail in Chapter 10. Recall ihaiATCase is inhibited by CTP, the final product ofpyrimidine biosynthesis, and stimulated by ATP. Carbamoyl phosphate synthetase is a site of feedback inhibition in both prokaryotes and eukaryotes. 

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). 

Regulation of P.b. In E. coli carbamoyl phosphate synthetase is activated by the purine nucleotides, IMP and XMP, and it is inhibited by the pyrimidine nucleotides, UMP and UDP. The key control point is the synthesis of N-carbamoylaspartic acid, catalysed by aspartate carbamoyl-transferase (aspartate trans-carbamylase, EC 2.1.3.2). In E. coli and Aerobacter aerogenes this enzyme is inhibited by CTP, and the inhibition is prevented by ATP. In Pseudomonas fluor-escens, the enzyme is inhibited by UTP, whereas in higher plants the regulatory inhibitor is UMP. 

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