Purine nucleotides, interconversion

The de novo synthesis of inosinic acid The salvage pathways Purine nucleotide interconversions Other enzymes... 

The elucidation of the steps by which cells synthesize inosinic acid de novo for purine nucleotide interconversions is largely due to the brilliant work of Buchanan and his co-workers [65]. The biosynthetic scheme as it is currently envisioned is shown in Figure 2.4. 

Thioguanine (6-TG) also inhibits several enzymes in the de novo purine nucleotide biosynthetic pathway. Various metabolic lesions result, including inhibition of purine nucleotide interconversion decrease in intracellular levels of guanine nucleotides, which leads to inhibition of glycoprotein synthesis interference with the formation of DNA and RNA and incorporation of thiopurine nucleotides into both DNA and RNA. 6-TG has a synergistic action when used together with cytarabine in the treatment of adult acute leukemia. 

Thioguanine has multiple metabolic effects. Its tumor inhibitory properties may be due to one or more of its effects on feedback inhibition of de novo purine synthesis inhibition of purine nucleotide interconversions or incorporation into DNA and RNA. The net conseqnence of its actions is a sequential blockade of the synthesis and utilization of the purine nucleotides. 

The dual role of adenylosuccinate synthetase in purine nucleotide interconversions and in de novo AMP biosynthesis complicates studies of its regulation. There is evidence suggesting that in wild-type cells, both the de novo and the salvage pathways play an active role in the maintenance of appropriate ATP/GTP ratios (78). 

Guanylate reductase, which deaminates this nucleotide, catalyzes a reductive, rather than hydrolytic, deamination and has been discussed in Chapter 9. Like adenylate deaminase, it has a catabolic role and also functions in purine nucleotide interconversion. A guanosine deaminase has recently been identified in a pseudomonad (13), but it is not known to occur in animal cells. 

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]. 

In Fig. 1 various targets of some important cytostatic agents are depicted. Their main mechanisms of action can be briefly summarized as follows. Pentostatin blocks purine nucleotides by inhibiting adenosine deaminase. 6-Mercaptopurine and 6-thioguanine inhibit purine ring biosynthesis and they inhibit nucleotide interconversions. Methotrexate by inhibiting dihydrofolate reduction blocks thymidine monophosphate and purine synthesis. 5-Fluorouracil also blocks thymidine monophosphate synthesis. Dactinomycin, daunorubicin, doxorubicin and mitoxantrone intercalate with DNA and inhibit RNA synthesis. L-asparaginase deaminates... 

Le Floc h, F., Lafleuriel, J., and Guillot, A., Interconversion of purine nucleotides in Jerusalem artichoke shoots, Plant Sci. Lett., 27, 309-316, 1982. 

The salvage pathway does not involve the formation of new heterocyclic bases but permits variation according to demand of the state of the base (B), i.e. whether at the nucleoside (N), or nucleoside mono- (NMP), di- (NDP) or tri- (NTP) phosphate level. The major enzymes and routes available (Scheme 158) all operate with either ribose or 2-deoxyribose derivatives except for the phosphoribosyl transferases. Several enzymes involved in the biosynthesis of purine nucleotides or in interconversion reactions, e.g. adenosine deaminase, have been assayed using a method which is based on the formation of hydrogen peroxide with xanthine oxidase as a coupling enzyme (81CPB426). 

Since the two isozymes of adenylosuccinate synthetase differ so markedly, changes in the relative amounts of the two could drastically affect the regulation of the reaction they catalyze, and therefore the direction of purine nucleotide metabolism. Determination of this ratio could be a useful indicator of the relative importance of the biosynthetic and the cyclic aspects of the adenine nucleotide interconversion pathway in different tissues or under different metabolic conditions. 

The regulation of mammalian adenylosuccinate synthetase is complicated. It is dependent on the isozyme content and levels in a given tissue as well as the effects of substrate and product levels. The two isozymes may have different metabolic roles either in AMP biosynthesis and interconversion, or in the functions of the purine nucleotide cycle. Most studies have considered kinetic parameters for the isolated enzyme and in only a few instances has regulation been studied in vivo. Sufficient information is available concerning the regulation of the basic isozyme in muscle to consider that enzyme in detail. Factors controlling the acidic isozyme are less clearly defined. 

Several of the enzymes which catalyze the interconversion of the 114-folate coenzymes are inhibited or repressed by purine nucleotides. Thus, 5,10-methylene H4-folate dehydrogenase is inhibited by ATP, GTP, and ITP (66), and 5,10-methylene H4-folate reductase is inhibited by S-adenosylmethionine (67). 10-Formyl H4-folate synthetase is repressed in cells grown in the presence of purines (68). 

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. 

The actual physiological role of these stimulatory and inhibitory effects of purine nucleotides on the enzymes of purine ribonucleoside interconversion is very difficult to assess, and little is knovm about the matter at the present time. Inhibition of inosinate dehydrogenase by metabolites of guanine (S6) and inhibition of guanylate reductase by metabolites of adenine (41) appear to occur in some bacteria supplied with these bases. 

Fig. 1. Interconversions of purine nucleotides. The wavy line indicates activation the broken lines indicate inhibition. PR-ATP, N-l- phosphoribosyl-ATP S-AMP, adenylo-succinic acid AICAR, phosphoribosyl-aminoimidazolecarboxamide.

Previous studies in our laboratory have demonstrated the pathways for purine salvage and interconversion associated with purine nucleotide synthesis in falciparum infected erythrocytes (PRBC) (5). The malaria parasite cannot synthesize purines de novo (4). Hypoxanthine appears to be the preferred substrate for synthesis of both adenosine and guanosine nucleotides, although adenine and guanine can be used (5). Figure 1 shows a representative nucleotide profile for PCA extracted PRBC following incubation with ( H) hypoxanthine. Label from hypoxanthine was incorporated into IMP and then into both adenylates and guanylates. 

PURINE NUCLEOTIDE SYNTHESIS, INTERCONVERSION AND CATABOLISM IN HUMAN LEUKOCYTES... 

These one-carbon groups, which are required for the synthesis of purines, thymidine nucleotides and for the interconversion some amino acids, are attached to THF at nitrogen-5 (N5), nitrogen-10 (N10) or both N5and N10. Active forms of folate are derived metabolically from THF so a deficiency of the parent compound will affect a number of pathways which use any form of THF. 

Molecular mechanics and dynamics studies of metal-nucleotide and metal-DNA interactions to date have been limited almost exclusively to modeling the interactions involving platinum-based anticancer drugs. As with metal-amino-acid complexes, there have been surprisingly few molecular mechanics studies of simple metal-nucleotide complexes that provide a means of deriving reliable force field parameters. A study of bis(purine)diamine-platinum(II) complexes successfully reproduced the structures of such complexes and demonstrated how steric factors influenced the barriers to rotation about the Pt(II)-N(purine) coordinate bonds and interconversion of the head-to-head (HTH) to head-to-tail (HTT) isomers (Fig. 12.4)[2011. In the process, force field parameters for the Pt(II)/nucleotide interactions were developed. A promising new approach involving the use of ab-initio calculations to calculate force constants has been applied to the interaction between Pt(II) and adenine[202]. 

---