Pyrimidine aspartate transcarbamoylase

Aspartate transcarbamoylase (ATCase), the catalyst for the first reaction unique to pyrimidine biosynthesis (Figure 34-7), is feedback-inhibited by cytidine tri-... 

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. 

Allosteric enzymes are generally larger and more complex than nonallosteric enzymes. Most have two or more subunits. Aspartate transcarbamoylase, which catalyzes an early reaction in the biosynthesis of pyrimidine nucleotides (see Fig. 22-36), has 12 polypeptide chains organized into catalytic and regulatory subunits. Figure 6-27 shows the quaternary structure of this enzyme, deduced from x-ray analysis. 

Regulation of the rate of pyrimidine nucleotide synthesis in bacteria occurs in large part through aspartate transcarbamoylase (ATCase), which catalyzes the first reaction in the sequence and is inhibited by CTP, the end product of the sequence (Fig. 22-36). The bacterial ATCase molecule consists of six catalytic subunits and six regulatory subunits (see Fig. 6-27). The catalytic subunits bind the substrate molecules, and the allosteric subunits bind the allosteric inhibitor, CTP. The entire ATCase molecule, as well as its subunits, exists in two conformations, active and inactive. When CTP is... 

The second step in pyrimidine synthesis is the formation of car-bamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed hydrolytically by dihydroorotase. Thi resulting dihydroorotate is oxidized to produce orotic acid (onotate, Figure 22.21). The enzyme that produces orotate, dihydroorotate dehydrogenase, is located inside the mitochondria. All other reactions in pyrimidine biosynthesis are cytosolic. [Note The first three enzymes in this pathway (CPS II, aspartate transcarbamoylase, and dihydroorotase) are all domains of the same polypeptide chain. (See k p. 19 for a discussion of domains.) This is an example of a multifunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound.]... 

A plot of VQ against [S] for an allosteric enzyme gives a sigmoidal-shaped curve. Allosteric enzymes often have more than one active site which co-operatively bind substrate molecules, such that the binding of substrate at one active site induces a conformational change in the enzyme that alters the affinity of the other active sites for substrate. Allosteric enzymes are often multi-subunit proteins, with an active site on each subunit. In addition, allosteric enzymes may be controlled by effector molecules (activators or inhibitors) that bind to a site other than the active site and alter the rate of enzyme activity. Aspartate transcarbamoylase is an allosteric enzyme that catalyzes the committed step in pyrimidine biosynthesis. This enzyme consists of six catalytic subunits each with an active site and six regulatory subunits to which the allosteric effectors cytosine triphosphate (CTP) and ATP bind. Aspartate transcarbamoylase is feedback-inhibited by the end-product of the pathway, CTP, which acts as an allosteric inhibitor. In contrast, ATP an intermediate earlier in the pathway, acts as an allosteric activator. 

Aspartate transcarbamoylase (aspartate carbamoyltransferase ATCase), a key enzyme in pyrimidine biosynthesis (see Topic FI), provides a good example of allosteric regulation. ATCase catalyzes the formation of N-carbamoylaspar-tate from aspartate and carbamoyl phosphate, and is the committed step in pyrimidine biosynthesis (Fig. 2). The binding of the two substrates aspartate and carbamoyl phosphate is cooperative, as shown by the sigmoidal curve of V0 against substrate concentration (Fig. 3). 

Fig. 2. Formation of N-carbamoylaspartate by aspartate transcarbamoylase (ATCase) is the committed step in pyrimidine biosynthesis and a key control point.

Unlike in purine biosynthesis, the pyrimidine ring is synthesized before it is conjugated to PRPP. The first reaction is the conjugation of carbamoyl phosphate and aspartate to make N-carbamoylaspartate. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm, in contrast to the carbamoyl phosphate used in urea synthesis, which is made in the mitochondrion. The enzyme that carries out the reaction is aspartate transcarbamoylase, an enzyme that is closely regulated. 

Pyrimidine synthesis is controlled at the first committed step. ATP stimulates the aspartate transcarbamoylase reaction, while CTP inhibits it. CTP is a feedback inhibitor of the pathway, and ATP is a feed-forward activator. This regulation ensures that a balanced supply of purines and pyrimidines exists for RNA and 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. 

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. 

There are two multifunctional proteins in the pathway for de novo biosynthesis of pyrimidine nucleotides. A trifunctional protein, called dihydroorotate synthetase (or CAD, where the letters are the initials of the three enzymatic activities), catalyzes reactions 1, 2 and 3 of the pathway (HCC>5"- CAP— CA-asp—> DHO Fig. 15-15). The enzymatic activities of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase, are contained in discrete globular domains of a single polypeptide chain of 243 kDa, where they are covalently connected by segments of polypeptide chain whch are susceptible to digestion by proteases such as trypsin. A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate— OMP—> UMP Fig. 15-15). Two enzymatic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa which associates as a dimer. 

Fig. 15-15 The de novo pyrimidine biosynthetic pathway. CAP, carbamoyl phosphate CA-asp, /V-carbamoyl-L-aspartate DHO, L-dihydroorotate Oro, orotate OMP, orotidine 5 -monophosphate. Enzymes (1) carbamoyl phosphate synthetase II (2) aspartate transcarbamoylase (3) dihydroorotase, (4) dihydroorotate dehydrogenase (5) orotate phosphoribosyltransferase (6) OMP decarboxylase (7) nucleoside monophosphate kinase (8) nucleoside diphosphate kinase (9) CTP synthetase.

Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines, bases that are components of nucleic acids. The reaction catalyzed by this enzyme is the condensation of aspartate and carbamoyl phosphate to form A-carbamoylaspartate and orthophosphate (Figure 10.1). ATCase catalyzes the committed step in the pathway that will ultimately yield pyrimidine nucleotides such as cytidine triphosphate (CTP). How is this enzyme regulated to generate precisely the amount of CTP needed by the cell ... 

Figure 10.1. ATCase Reaction. Aspartate transcarbamoylase catalyzes the committed step, the condensation of aspartate and carbamoyl phosphate to form A-carhamoylaspartate, in pyrimidine synthesis.

Figure 10.2. CTP luhibits ATCase. Cytidine triphosphate, an end product of the pyrimidine synthesis pathway, inhibits aspartate transcarbamoylase despite having little structural similarity to reactants or products.

We have previously encountered carbamoyl phosphate as a sub- strate for aspartate transcarbamoylase, the enzyme that catalyzes the first step in pyrimidine biosynthesis (Section 10.1). Carbamoyl phosphate synthetase... 

What about the other enzymes in the urea cycle Ornithine transcarbamoylase is homologous to aspartate transcarbamoylase and the structures of their catalytic subunits are quite similar (Figure 23.18). Thus, two consecutive steps in the pyrimidine biosynthetic pathway were adapted for urea synthesis. The next step in the urea cycle is the addition of aspartate to citrulline to form argininosuccinate, and the subsequent step is the removal of fumarate. These two steps together accomplish the net addition of an amino group to citrulline to form arginine. Remarkably, these steps are analogous to two consecutive steps in the purine biosynthetic pathway (Section 25.2 3). 

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 pyrimidine ring is assembled first and then linked to ribose phosphate to form a pyrimidine nucleotide. PRPP is the donor of the ribose phosphate moiety. The synthesis of the pyrimidine ring starts with the formation of carbamoylaspartate from carbamoyl phosphate and aspartate, a reaction catalyzed by aspartate transcarbamoylase. Dehydration, cyclization, and oxidation yield orotate, which reacts with PRPP to give orotidylate. Decarboxylation of this pyrimidine nucleotide yields UMP. CTP is then formed by the amination of UTP. 

Pyrimidine biosynthesis in E. coli is regulated by the feedback inhibition of aspartate transcarbamoylase, the enzyme that catalyzes the committed step. CTP inhibits and ATP stimulates this enzyme. The feedback inhibition of glutamine-PRPP amidotransferase by purine nucleotides is important in regulating their biosynthesis. 

Regulation of the de novo pathway is complex. In prokaryotic cells, aspartate transcarbamoylase, an allosteric protein, is inhibited by the end products of pyrimidine... 

A 4-year-old girl presents in the clinic with megaloblastic anemia and failure to thrive. Blood chemistries reveal orotic aciduria. Enzyme measurements of white blood cells reveal a deficiency of the pyrimidine biosynthesis enzyme orotate phosphoribosyltransferase and abnormally high activity of the enzyme aspartate transcarbamoylase. Which one of the following treatments will reverse all symptoms if carried out chronically ... 

The answer is e. (Murray, pp 375-401. Scriver, pp 2663-2704. Sack, pp 121-138. Wilson, pp 287—320.) Orotic aciduria is the buildup of orotic acid due to a deficiency in one or both of the enzymes that convert it to UMP Either orotate phosphoribosyltransferase and orotidylate decarboxylase are both defective, or the decarboxylase alone is defective. UMP is the precursor of UTP, CTP, and TMP All of these end products normally act in some way to feedback-inhibit the initial reactions of pyrimidine synthesis. Specifically, the lack of CTP inhibition allows aspartate transcarbamoylase to remain highly active and ultimately results in a buildup of orotic acid and the resultant orotic aciduria. The lack of CTP, TMP, and UTP leads to a decreased erythrocyte formation and megaloblastic anemia. Uridine treatment is effective because uridine can easily be converted to UMP by omnipresent tissue kinases, thus allowing UTP, CTP, and TMP to be synthesized and feedback-inhibit further orotic acid production. 

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. 

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