Purine bases, nucleotide formation from

The mdstence of a feedback control mechanism for purine biosynthesis was suggested by the observation that unlabeled purines inhibited purine theris de novo from labeled precursors in several systems (SB, 1B4,445-447). Initially, it was postulated that the feedback inhibition by preformed purines may have involved a shuntii of PRPP from the de novo route of purine biosyntheris and utilization of PRPP for nucleotide formation from the purine base and PRPP. PRPP was an obligatory intermediate in the synthesis of purines de novo. Since the nucleotide was still formed albeit by a different route, this would not be considered a feedback mechanism. It is probable, however, that the accumulation of the newly-formed nucleotide from PRPP and base inhibited the de novo route. 

The determination of purine nucleotide formation from purine bases in the presence of ribose-1-phosphate and deoxyri-bos0-1-phosphate was carried out using the same reaction mixture as for the purine-PRT activities, but instead of PRPP,... 

Nucleotide formation from free purine bases is dependent on the availability of phosphoribosylpyrophos-phate (PRPP), the common substrate for both HGPRT and adenine phosphoribosyltransferase (APRT) (4). Vhereas human platelets do contain PRPP, the activity of PRPP synthetase in these cells has not been reported thus far (2). 

In mammals specific enzymes for converting purine bases to nucleotides are present in many organs, and in heart muscle this may be the main source of purine nucleotides. The most important of these enzymes is hypoxanthine-guanine phosphoribosyltransferase, which catalyzes the formation of IMP from hypoxanthine and GMP from guanine ... 

All biosynthetic pathways are under regulatory control by key allosteric enzymes that are influenced by the end products of the pathways. For example, the first step in the pathway for purine biosynthesis is inhibited in a concerted fashion by nucleotides of either adenine or guanine. In addition, the nucleoside monophosphate of each of these bases inhibits its own formation from inosine monophosphate (IMP). On the other hand, adenine nucleotides stimulate the conversion of IMP into GMP, and GTP is needed for AMP formation. 

Purine nucleotides can be synthesized in the organism from relatively simple building blocks ribose, phosphate, glycine, formate, aspartate, glutamine, and C02. The origin of each purine base component is summarized in Figure 10.5,... 

Free purine bases, derived from the turnover of nucleotides or from the diet, can be attached to PRPP to form purine nucleoside monophosphates, in a reaction analogous to the formation of orotidylate. Two salvage enzymes with different specificities recover purine bases. Adenine phosphorihosyltransferase catalyzes the formation of adenylate... 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

The inhibitory effects of allopurinol and oxipurinol on purine and pyrimidine biosynthesis in human fibroblasts could be explained by formation of their respective ribonucleotides either by HGPRTase (allopurinol) or OPRTase (oxipurinol). The effects of allopurinol-1-ribonucleoside are hardly to be explained. Theoretically the allopurinol-1-ribonucleoside can be converted either to the free base (catalyzed by a purine nucleoside phosphorylase) or to allopurinol-1-ribonucleotide (catalyzed by a nucleoside kinase). According to Utter et al. (4) there seems however to be a lack of kinases in mammals which effectively phosphorylate inosine. Furthermore, indirect experiments of Elion et al. (5), where no detectable nucleotide formation or incorporation into nucleic acids was observed in vivo with Hc-allopurinol would support this. Nevertheless, our results would claim for a direot conversion of allopurin-ol-1-ribonucleoside to allopurinol-1-ribonucleotide in human fibroblast, at least at doses from 10 5 -to 10-3 M. Otherwise we would have to explain the inhibitory effects of allopurinol-1-ribonucleoside by direct influences on purine synthesis, for the other possibility theoretical-... 

Since there has been no evidence presented to support the hypothesis that free adenine can be formed de novo in biological systems from small molecule precursors, and furthermore, since purines have never been reported to have been essential dietary additions, the formation of nucleotides from free purines may be looked upon as a minor biosynthetic pathway. Undoubtedly, there is some utilization of free purines which are derived from the intestinal tract as well as from catabolic events within the cell. The term salvage pathway has been aptly applied to the reactions utilizing free bases for nucleic acid synthesis (206). 

In proliferating cells purine nucleotides - necessary for DNA- and RNA-synthesis - can be formed by incorporation of small precursors (de novo pathway) and by utilisation of free purin bases (salvage pathway). The most important reactions of both metabolic pathways are demonstrated in figure 1. The first reaction of the de-novo pathway - the formation of phosphoribosylamine from glutamine and phosphoribosyIpyrophosphate (PRPP) by the enzyme PRPPamidotransferase - is the target of a negative feed back control mechanism by the endproducts IMP/GMP and AMP (1). 

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,... 

The base of a nucleotide is joined covalently (at N-l of pyrimidines and N-9 of purines) in an iV-/3-glycosyl bond to the 1 carbon of the pentose, and the phosphate is esterified to the 5 carbon. The AT-j3-glycosyl bond is formed by removal of the elements of water (a hydroxyl group from the pentose and hydrogen from the base), as in O-glycosidic bond formation (see Fig. 7-31). 

PRPP is the activated intermediate in the synthesis of phosphoribosylamine in the de novo pathway of purine formation of purine nucleotides from free bases by the salvage pathway of orotidylate in the formation of pyrimidines of nicotinate ribonucleotide of phosphoribosyl ATP in the pathway leading to histidine and of phosphoribosylanthranilate in the pathway leading to tryptophan. 

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