Pyrimidine phosphoribosyltransferases

In the pyrimidines, a mammalian erythrocyte enzyme, pyrimidine phosphoribosyltransferase, converts uracil, orotic acid, and thymine into nucleotides as follows ... 

At this stage, orotate couples to ribose, in the form of 5-phosphoribosyl-l-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide bases. PRPP is synthesized from ribose-5-phosphate, formed by the pentose phosphate pathway, by the addition of pyrophosphate from ATP. Orotate reacts with PRPP to form orotidylate, a pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The enzyme that catalyzes this addition, pyrimidine phosphoribosyltransferase, is homologous to a number of other phosphoribosyltransferases that add different groups to PRPP to form the other nucleotides. Orotidylate is then decarboxylated to form uridylate (IMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. 

Similar salvage pathways exist for pyrimidines. Pyrimidine phosphoribosyltransferase will reconnect uracil, but not cytosine, to PRPP. 

Pyrimidine ribonucleotides, like those of purines, may be synthesized de novo from amino acids and other small molecules (Chapter 11). Preformed pyrimidine bases and their ribonucleoside derivatives, derived from the diet of animals or found in the environment of cells, may be converted to ribonucleotides via nucleoside phosphorylases and nucleoside kinases. In some cells a more direct pyrimidine phosphoribosyltransferase pathway has also been recognized (Chapter 12). Ribonucleotides are catabolized by dephosphorylation, deamination, and cleavage of the glycosidic bond, to uracil. Uracil may be either oxidatively or reductively cleaved, depending on the organism involved, and can be converted to CO and NH (Chapter 13). 

A pyrimidine phosphoribosyltransferase activity with a broader specificity than the yeast enzyme has been demonstrated in animal tissues. Highly purified preparations from calf thymus (15) and beef erythrocytes (16) accepted orotate and 5-fluorouracil as substrates. Uracil phosphoribosyltransferase activity has also been demonstrated in extracts from mouse leukemia cells. Fluorouracil is a better substrate for this enzyme than uracil at pH 7.5, possibly because the acid dissociation constant for the analogue (pif 8.15) is higher than that of uracil (pK, 9.45) (17). This reasoning would suggest that the anionic form of the substrate might be the species required by the enzyme. This enzyme has been implicated in the... 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

Many yeasts are inhibited by 5-fluorocytosine and a block in the synthesis of 5-fluorouridylic acid by loss of cytosine deaminase or of nracil phosphoribosyltransferase is sufficient to cause resistance. Mntational loss of pyrimidine salvage enzymes has been frequently observed. 

The enzymatic conversion of many analogues of the naturally occurring purines directly to their biologically active form, the ribonucleotides, in vivo [5, 8, 10, 13, 39] underlines the importance of these enzymes to the drug action of this class of compounds. 2-Aminoadenine (2, 6-diaminopurine, I) [107], 2-fluoroadenine (II) [108], 4-aminopyrazolo [3, 4-d] pyrimidine (VIll) [109]. and 2- and 8-aza-adenine (IX and X) [ 110, 111] have all been shown to be substrates for the adenine phosphoribosyltransferase [J12, 113]. Extensive studies on the metabolism of 2-aminoadenine (I) in E. coli [114, 115], L cells [116], and mice [117] have also shown its conversion by this enzyme to the ribonucleotide. 

Fluoroadenine, 2-chloroadenine, 2-aminoadenine, 2- and 8-aza-adenines, 4-aminopyra7.olo[3, 4-d] pyrimidine and 6-methylpurine are converted to their ribonucleosidesby adenosine phosphoribosyltransferase,and their ribonucleosides are converted to the ribonucleotides by adenosine kinase most of the ribonucleotides are then converted to the di- and triphosphates. A -Aminoadenine, A -hydroxyadenine, A -methyladenine, purine, 7-deaza-adenine, and 7-amino-pyrazolo[4, 3-d] pyrimidine are either not substrates or are very poor substrates for the phosphoribosyltransferase, but their ribonucleosides are excellent substrates for the kinase. The ribonucleotides of purine, 7-deaza-adenine and... 

Table 7.1.4 Concentration range of purine and pyrimidine metabolites in urine (pmol/mmol creatinine) from patients. ADA Adenosine deaminase, APRT adenine phosphoribosyltransferase, ASA adenylosuccinate lyase, DHP dihydropyrimidinase, DPD dihydropyrimidine dehydrogenase, HGPRT hypoxanthine-guanine phosphoribosyltransferase, PNP purine nucleoside phosphorylase, TP thymidine phosphorylase, UMPS uridine monophosphate synthase, / -UP fi-ureidopropionase... 

Free purine and pyrimidine bases are constantly released in cells during the metabolic degradation of nucleotides. Free purines are in large part salvaged and reused to make nucleotides, in a pathway much simpler than the de novo synthesis of purine nucleotides described earlier. One of the primary salvage pathways consists of a single reaction catalyzed by adenosine phosphoribosyltransferase, in which free adenine reacts with PRPP to yield the corresponding adenine nucleotide ... 

Free guanine and hypo xanthine (the deamination product of adenine Fig. 22-45) are salvaged in the same way by hypoxanthine-guanine phosphoribosyltransferase. A similar salvage pathway exists for pyrimidine bases in microorganisms, and possibly in mammals. 

Just as orotic acid is converted to a ribonucleotide in step e of Fig. 25-14, other free pyrimidine and purine bases can react with PRPP to give monoribonucleotides plus PP . The reversible reactions, which are catalyzed by phosphoribosyltransferases (ribonucleotide pyrophosphorylases), are important components of the salvage pathways by which purine and pyrimidine bases freed by the degradation of nucleic acids are recycled.273 However, thymine is usually not reused. Thymine will react with deoxribose 1-P to form thymidine plus inorganic phosphate (thymidine phosphorylase), and thymidine is rapidly... 

In the third step, the pyrimidine ring is closed by dihy-droorotase to form 1-dihydroorotate. Dihydroorotate is then oxidized to orotate by dihydroorotate dehydrogenase. This flavoprotein in some organisms contains FMN and in others both FMN and FAD. It also contains nonheme iron and sulfur. In eukaryotes it is a lipoprotein associated with the inner membrane of the mitochondria. In the final two steps of the pathway, orotate phosphoribosyltransferase yields orotidine-5 -phosphate (OMP), and a specific decarboxylase then produces UMP. 

Low activities of orotidine phosphate decarboxylase and (usually) orotate phosphoribosyltransferase are associated with a genetic disease in children that is characterized by abnormal growth, megaloblastic anemia, and the excretion of large amounts of orotate. When affected children are fed a pyrimidine nucleoside, usually uridine, the anemia decreases and the excretion of orotate diminishes. A likely explanation for the improvement is that the ingested uridine is phosphorylated to UMP, which is then converted to other pyrimidine nucleotides so that nucleic acid and protein synthesis can resume. In addition, the increased intracellular concentrations of pyrimidine nucleotides inhibit carbamoyl phosphate synthase, the first enzyme in the. naibwav of aro-tate synthesis. 

In eukaryotes, carbamoyl phosphate synthase is inhibited by pyrimidine nucleotides and stimulated by purine nucleotides it appears to be the most important site of feedback inhibition of pyrimidine nucleotide biosynthesis in mammalian tissues. It has been suggested that under some conditions, orotate phosphoribosyltransferase may be a regulatory site as well. 

Such conversions in mammals are not very efficient, however, with the exception of, perhaps, the orotate phosphoribosyltransferase, which is a component of the pyrimidine nucleotide biosynthetic pathway (Figure 10.9). A very active uracil phosphoribosyltransferase has been isolated from microorganisms. It converts uracil to UMP using PRPP. 

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.

The metabolic interrelationship between mitochondrial carbamoyl phosphate synthesis to urea formation and to cytosolic carbamoyl phosphate channeled into pyrimidine biosynthesis. In ornithine transcarbamoyiase (OTC) deficiency, mitochondrial carbamoyl phosphate diffuses into the cytosol and stimulates pyrimidine biosynthesis, leading to orotidinuria. Administration of allopurinol augments orotidinuria by increasing the flux in the pyrimidine biosynthetic pathway. CPS = Carbamoyl phosphate synthase, AT = aspartate transcarbamoyiase, D = dihydroorotase, DH = dihydroorotate dehydrogenase, OPRT = orotate phosphoribosyltransferase, XO = xanthine oxida.se,... 

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. 

Enzymes that act on PRPP include Phosphoribosyltransferases (salvage synthesis and de novo synthesis of pyrimidines), PRPP amidotransferase... 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate —> OMP — UMP Fig. 14-7). Two enzymic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa that associates as a dimer. 

The de novo synthesis of pyrimidines and pnrines, particnlarly pnrines, is energetically expensive so most (-80%) of the pnrines and pyrimidines obtained from the degradation of nncleic acids, particnlarly RNA, are salvaged for rense. Hrnnan cells contain three phosphoribosyltransferases (PRTases) that convert preformed... 

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