De novo synthesis of purine nucleotides

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 ... 

Mechanism of overproduction of purine nucleotides in the congenital deficiency of hypoxanthine-guanine phosphoribosyltransferase. The loss of the transferase prevents the recycling of hypoxanthine and guanine. This increases uric acid production as well as the de novo synthesis of purine nucleotides. 

A highly unusual feature of DHFR in Apicomplexa and Kinetoplastida is its association with thymidylate synthase in the same protein. DHFR activity is always located at the amino terminal portion, while the thymidylate synthase activity resides in the carboxyl terminal. The two enzyme functions do not appear to be interdependent eg, the DHFR portion of the P falciparum enzyme molecule was found to function normally in the absence of the thymidylate synthase portion. It is likely that since the protozoan parasites do not perform de novo synthesis of purine nucleotides, the primary function of the tetrahydrofolate produced by DHFR is to provide 5,10-methylenetetrahydrofolate only for the thymidylate synthase-catalyzed reaction. Physical association of the two enzymes may improve efficiency of TMP synthesis. If an effective means of disrupting the coordination between the two activities can be developed, this bifunctional protein may qualify as a target for antiparasitic therapy. 

Scheme 12-6 De novo synthesis of purine nucleotides (simplified).

The six one-carbon substituents of tetrahydrofolate. The oxidation state is the same in the one-carbon moiety of N -formyl, N -formyl, and N, N" -methenyl FH4. N -FormyI FH4 is required for de novo synthesis of purine nucleotides, whereas N, N -methylene FH4 is needed for formation of thymidilic acid. 

The purine ring is assembled from a variety of precursors glutamine, glycine, aspartate, N formyltetrahydrofolate, and (XT. The committed step in the de novo synthesis of purine nucleotides is the formation of 5-phosphoribosyIamine from PRPP and glutamine. The purine ring is assembled on ribose phosphate, in contrast with the de novo synthesis of pyrimidine nucleotides. The addition of glycine,... 

PURINE NUCLEOTIDES The de novo synthesis of purine nucleotides begins with the formation of 5-phospho-a-D-ribosyl- 1-pyrophosphate (PRPP) catalyzed by ribose-5-phosphate pyrophosphokinase (PRPP synthetase). 

No evidence for the presence of a known enzyme abnormality causing purine overproduction could be obtained. The erythrocyte activity of hypoxanthine-guanine phos-phoribosyltransferase (HGPRT), of adenine phosphoribosyltransferase (APRT), and of phosphoribosylpyrophosphate (PRPP) synthetase were all in the normal range. Erythrocyte PRPP generation, as well as the acitivity of the pentose phosphate pathway was also normal (Table 1). In addition, the rate of de novo synthesis of purine nucleotides in cultured skin fibroblasts from the patient was found to be normi. 

Phosphoribosylpyrophosphate (PRPP) synthetase (E.C. 2.7.6.1) catalyzes the formation of PRPP from ribose-5-phosphate and ATP in the presence of Mg and inorganic phosphate. The product, PRPP, is a substrate of the first rate limiting step of the de novo synthesis of purine nucleotides and its availability has been shown to regulate this pathway in human tissue (l). A superactive mutant erythrocyte PRPP synthetase with decreased sensitivity to feedback inhibition has recently been found by us in a gouty family (2,3). 

Since pentoses and phosphates were not included in the HCN-NH4OH mixtures one could not expect the formation of nucleosides or nucleoside phosphates in such mixtures, as in the biochemical de novo synthesis of purine nucleotides, even though an adenine derivative of possible nucleosidic nature was detected in one of our early experiments. Alternate biochemical routes also exist (salvage pathways) for the synthesis of nucleosides from the preformed bases. The latter general approach has been followed in the prebiotic synthesis of purine nucleosides, and the former has been used in the synthesis of pyrimidine nucleosides and nucleotides. 

The a- and jS-D-arabinofuranosyl and a- and jS-o-xylofuranosyl nucleotide analogues [(493) and (494), respectively] and the 2-bromo derivative (495) of 5-amino-l-j3-D-ribofuranosylimidazole-4-carboxyIic acid 5 -phosphate (a central intermediate in the de novo synthesis of purine nucleotides) and its a-anomer have been synthesized from 2,3,5-tri-0-benzoyl-aj8-D-arabinofuranosyl azide, 3,5-0-isopropylidene-aj8-D-xylofuranosylamine, and ethyl 5-amino-l-a- and - -d-ribofuranosylimidazole-4-carboxylates, respectively. The jS-anomers of (493)—... 

In a similar way the purine antimetabolite 6-mercapto-purine (6-MP)/ after conversion to the monophosphoribo-tide, acts as an inhibitor of this enzyme (9.10). There have been described other sites of action of 6-MP (4-7) which are marked by arrows in figure 1. We have previously demonstrated the inhibition of the enzymic formate activation (tetrahydrofolate formylase) in leukemic cells by 6-MP (12). inspite of a rather high inhibitory concentration of 6-MP between 10 and 10 M, this inhibition has some practical clinical implications for the treatment of acute leukemia/ as it is detected in sensitive leukemic cells only. In accordance with reports of several authors (8,9/10) we have postulated/ that 6-MP has to be converted into 6-thioinosinic acid for exerting its inhibitory effect on the de novo synthesis of purine-nucleotides. On the other hand DAVIDSON and WINTER have shown that both 6-MP sensitive and resistant cells con-... 

The carbon atoms 2 and 8 in the purine ring of inosinic acid are derived from C1 units. The latter are transferred as activated formate to GAR and AICR as specific formate acceptors. Therefore we have studied the tetra-hydrofolate dependent activation of formate in relation to the netto de novo synthesis of purine nucleotides in cell-free extracts of normal and leukemic leukocytes. In addition, the conversion of exogenous purines to corresponding monophosphoribonucleotides by the specific purine-phosphoribosyItransferases was determined. The aim of these investigations was to study the effect of 6-MP on the formate activating system, which is important for the de novo synthesis of purine nucleotides, on the utilization of preformed purine bases and, in addition, the interaction of allopurinol with 6-MP,... 

For comparising the activity of the tetrahydrofolate formylase with the netto de novo synthesis of purine nucleotides incubations were performed with 14C-formate and the components necessary for the enzymic formate activation only and in a parallel experiment with additional KHCO3, glutamine,glycine and ribose-5-phosphate (12). Determinations of the tetrahydrofolate formylase activity and of... 

The tetrahydrofolate formylase was found in normal and leukemic leukocytes, the highest activities in immature blast cells of acute Leukemia (table 1). By determination of the 14C-formate incorporation a measurable netto de novo synthesis of purine nucleotides was only detected in immature leukemic cells. 

---