Purine ribonucleotide synthesis

N4. Nierlich, D. P., and Magasanik, B., Regulation of purine ribonucleotide synthesis by end product inhibition. J. Biol. Chem. 240, 358-365 (1965). 

PURINE RIBONUCLEOTIDE SYNTHESIS FROM PURINE BASES AND RIBONUC LEO SIDES... 

Enzymes of Purine Ribonucleotide Synthesis from Bases and Ribonucleosides... 

The two classes of nucleotide that must be synthesised are the pyrimidine and purine ribonucleotides for RNA synthesis and the deoxyribonucleotides for DNA synthesis. For the original sources of the nitrogen atoms in the bases of the pyrimidine and purine nucleotides, see Figure 20.7. The pathway for the synthesis of the pyrimidine nucleotides is... 

The effect of 6-mercaptopurine on the incorporation of a number of C-labelled compounds into soluble purine nucleotides and into RNA and DNA has been studied in leukemia L1210, Ehrlich ascites carcinoma, and solid sarcoma 180. At a level of 6-mercaptopurine that markedly inhibited the incorporation of formate and glycine, the utilization of adenine or 2-aminoadenine was not affected. There was no inhibition of the incorporation of 5(or 4)-aminoimidazole-4(5)-carboxamide (AIC) into adenine derivatives and no marked or consistent inhibition of its incorporation into guanine derivatives. The conversion of AIC to purines in ascites cells was not inhibited at levels of 6-mercaptopurine 8-20 times those that produced 50 per cent or greater inhibition of de novo synthesis [292]. Furthermore, AIC reverses the inhibition of growth of S180 cells (AH/5) in culture by 6-mercaptopurine [293]. These results suggest that in all these systems, in vitro and in vivo, the principal site at which 6-mercaptopurine inhibits nucleic acid biosynthesis is prior to the formation of AIC, and that the interconversion of purine ribonucleotides (see below) is not the primary site of action [292]. Presumably, this early step is the conversion of PRPP to 5-phosphoribosylamine inhibited allosterically by 6-mercaptopurine ribonucleotide (feedback inhibition is not observed in cells that cannot convert 6-mercaptopurine to its ribonucleotide [244]. 

The common pyrimidine ribonucleotides are cytidine 5 -monophosphate (CMP cytidylate) and uridine 5 -monophosphate (UMP uridylate), which contain the pyrimidines cytosine and uracil. De novo pyrimidine nucleotide biosynthesis (Fig. 22-36) proceeds in a somewhat different manner from purine nucleotide synthesis the six-membered pyrimidine ring is made first and then attached to ribose 5-phosphate. Required in this process is carbamoyl phosphate, also an intermediate in the urea cycle (see Fig. 18-10). However, as we noted... 

Overview of Nucleotide Metabolism Synthesis of Purine Ribonucleotides de Novo... 

Purine deoxyribonucleotides are derived primarily from the respective ribonucleotide (Fig. 6.2). Intracellular concentrations of deoxyribonucleotides are very low compared to ribonucleotides usually about 1% that of ribonucleotides. Synthesis of deoxyribonucleotides is by enzymatic reduction of ribonucleotide-diphosphates by ribonucleotide reductase. One enzyme catalyzes the conversion of both purine and pyrimidine ribonucleotides and is subject to a complex control mechanism in which an excess of one deoxyribonucleotide compound inhibits the reduction of other ribonucleotides. Whereas the levels of the other enzymes involved with purine and pyrimidine metabolism remain relatively constant through the cell cycle, ribonucleotide reductase level changes with the cell cycle. The concentration of ribonucleotide reductase is very low in the cell except during S-phase when DNA is synthesized. While enzymatic pathways, such as kinases, exist for the salvage of pre-existing deoxyribosyl compounds, nearly all cells depend on the reduction of ribonucleotides for their deoxyribonucleotide... 

Purine bases can be converted to ribonucleotides via phosphoribosyl-transferases PP-ribose-P provides the ribosyl phosphate moiety. Purine nucleosides can be phosphorylated by ATP-requiring nucleoside kinases to form the same ribonucleotides. Finally, the possibility also exists that purine bases are first converted to ribonucleosides via nucleoside phos-phorylase, and then to ribonucleotides by the above-mentioned kinases. These routes of ribonucleotide synthesis are summarized as follows ... 

The synthesis of adenylate from inosinate in bone marrow and microbial preparations, and of guanylate from inosinate in pigeon liver, bone marrow, and bacterial extracts were reported between 1955 and 1957 (see references 2, 6-10). In the course of these studies, two new purine ribonucleotides were identified and shown to be intermediates in these processes. 

The activities of the enzymes of purine ribonucleotide interconversion can be both stimulated and inhibited in a variety of ways, and these potential control mechanisms may function not only to regulate the synthesis of ATP and of GTP, but also to maintain a balance in the relative intracellular concentrations of these two nucleotides. 

Based on our understanding of fructose metabolism, we have examined four potential mechanisms to account for fructose-induced hyperuricemia in man. 1) Shift in the uric acid pool, 2) decreased renal clearance of uric acid, 3) increased purine synthesis novo by stimulating PP-ribose-P production,and 4) accelerated degradation of purine ribonucleotides. Our studies were designed to distinguish which of these mechanisms in man could account for the hyperuricemia observed after fructose infusion. 

This hyperuricemic effect of xylitol could be due in part- comparable to that of fructose- to an increased rate of purine synthesis de novo or an accelerated degradation of purine ribonucleotides. 

Since allopurinol has a half-life of only about 1-1/2 hours in man, due to its rapid oxidation, the level of allopurinol ribonucleotide in human liver is probably < 0.0001 mM. If one uses, as a first approximation, the values reported for the pigeon liver PRPP-amidotransferase, = 0.6 mM, [16] the levels of allopurinol ribonucleotide in liver are far below the amount required to inhibit this enz5mie appreciably. On the other hand, the levels of the natural purine ribonucleotides [9] lie much closer to the values for the pigeon liver enz3nne [16,17] or to the [I]0,5 values in human l3miphoblasts [18]. Hypoxanthine and xanthine levels are increased by the inhibition of xanthine oxidase produced by allopurinol and oxipurinol. The reutilization of these oxypurines is very efficient under these conditions [19,20]. This could result in feedback inhibition of novo synthesis through temporary increases in IMP and XMP, and subsequently AMP and GMP levels. This effect would be enhanced by the cooperative effect of AMP and GMP in the inhibition of PRPP - amidotransferase [17]. 

The study of the effect of purine ribonucleotides on PRA synthesis in cell free enzyme preparations of these two lines of lymphocytes revealed that PRA synthesis of preparations of HGPRT deficient Lesch-Nyhan cells were sensitive to inhibition by AMP, GMP and allopurinol ribonucleotide (Table 4). The degree of inhibition of PRA synthesis of preparations of normal lymphocytes by AMP, QIP and allopurinol ribonucleotide was similar to that of preparations of Lesch-Nyhan lymphocytes. There was, however, a significant difference between the effect of AMP on PRA synthesis from PRPP and glutamine compared with that on PRA synthesis from PRPP ammonia. PRA synthesis from PRPP and ammonia was significantly more sensitive to inhibition by AMP than was PRA synthesis from PRPP and glutamine. 

TABLE 4. EFFECT OF PURINE RIBONUCLEOTIDES ON PRA SYNTHESIS IN EXTRACTS OF LESCH-NYHAN AND NORMAL LYMPHOCYTES... 

Larsson, a. Enzymatic synthesis of deoxyribonucleotides. III. Reduction of purine ribonucleotides with an enzyme system from Escherichia coli B. J. Biol. Chem. 238, 3414 (1963). 

Fig. 3. The pathway of de novo purine ribonucleotide biosynthesis. The pathway includes the synthesis of PRPP, which is also used in the synthesis of pyrimidines, pyridine nucleotides, histidine, and tryptophan in plants. The enzymes catalyzing the numbered reactions are (1) PRPP synthetase, (2) PRPP amidotransferase, (3) GAR synthetase, (4) GAR transformylase, (5) FGAR amidotransferase, (6) AIR synthetase, (7) AIR carboxylase, (8) succino-AICAR synthetase, (9) adenylosuccinase, (10) AICAR transformylase, and (11) IMP cyclohydrolase.

As discussed in the previous section, the synthesis of ribose 5-phosphate must be quite high to provide the ribose 5-phosphate required for de novo purine ribonucleotide biosynthesis. Ribose 5-phosphate required for PRPP synthesis can be synthesized de novo via the oxidative or nonoxidative arms of the pentose phosphate pathway or by recycling of ribose released by the action of nucleotidases/nucleosidases (Fig. 5). The latter pathway requires ribose phosphotransferase (ribokinase), which has been detected in soybean and pea nodule extracts (Christensen and Jochimsen, 1983). The efficient recycling of ribose could eliminate the need for the continuous production of ribose 5-phosphate. Two enzymes of the oxidative branch of the pentose phosphate... 

Mercaptopurine (6-MP) is an oral purine analog that is converted to a ribonucleotide to inhibit purine synthesis. Mercaptopurine is converted into thiopurine nucleotides, which are catabolized by thiopurine S-methyltransferase (TPMT), which is subject to genetic polymorphisms and may cause severe myelosuppression. TPMT status may be assessed prior to therapy to reduce drug-induced morbidity and the costs of hospitalizations for neutropenic events. Mercaptopurine is poorly absorbed, with a time to peak concentration of 1 to 2 hours after an oral dose. The half-life is 21 minutes in pediatric patients and 47 minutes in adults. Mercaptopurine is used in the treatment of acute lymphocytic leukemia and chronic myelogenous leukemia. Significant side effects include myelosuppression, mild nausea, skin rash, and cholestasis. When allopurinol is used in combination with 6-MP, the dose of 6-MP must be reduced by 66% to 75% of the usual dose because allopurinol blocks the metabolism of 6-MP. 

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