Purine nucleotides steps

The pathways for thiamine biosynthesis have been elucidated only partiy. Thiamine pyrophosphate is made universally from the precursors 4-amino-5-hydroxymethyl-2-methylpytimidinepyrophosphate [841-01-0] (47) and 4-methyl-5-(2-hydroxyethyl)thiazolephosphate [3269-79-2] (48), but there appear to be different pathways ia the eadier steps. In bacteria, the early steps of the pyrimidine biosynthesis are same as those of purine nucleotide biosynthesis, 5-Aminoimidazole ribotide [41535-66-4] (AIR) (49) appears to be the sole and last common iatermediate ultimately the elements are suppHed by glycine, formate, and ribose. AIR is rearranged in a complex manner to the pyrimidine by an as-yet undetermined mechanism. In yeasts, the pathway to the pyrimidine is less well understood and maybe different (74—83) (Fig. 9). 

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. 

The first step of this sequence, which is not unique to de novo purine nucleotide biosynthesis, is the synthesis of 5-phosphoribosylpyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate. Phosphoribosyl-pyrophosphate synthetase, the enzyme that catalyses this reaction [278], is under feedback control by adenosine triphosphate [279]. Cordycepin interferes with thede novo pathway [229, 280, 281), and cordycepin triphosphate inhibits the synthesis of PRPP in extracts from Ehrlich ascites tumour cells [282]. Formycin [283], probably as the triphosphate, 9-0-D-xylofuranosyladenine [157] triphosphate, and decoyinine (LXXlll) [284-286] (p. 89) also inhibit the synthesis of PRPP in tumour cells, and this is held to be the blockade most important to their cytotoxic action. It has been suggested but not established that tubercidin (triphosphate) may also be an inhibitor of this reaction [193]. 

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]. 

Purine (left). The purine nucleotide guano-sine monophosphate (CMP, 1) is degraded in two steps—first to the guanosine and then to guanine (Gua). Guanine is converted by deamination into another purine base, xanthine. 

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

As the first committed step in the biosynthesis of AMP from IMP, AMPSase plays a central role in de novo purine nucleotide biosynthesis. A 6-phosphoryl-IMP intermediate appears to be formed during catalysis, and kinetic studies of E. coli AMPSase demonstrated that the substrates bind to the enzyme active sites randomly. With mammalian AMPSase, aspartate exhibits preferred binding to the E GTPTMP complex rather than to the free enzyme. Other kinetic data support the inference that Mg-aspartate complex formation occurs within the adenylosuccinate synthetase active site and that such a... 

Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine nucleotide synthesis and the relative rates of formation of the two end products, adenylate and guanylate (Fig. 22-35). The first mechanism is exerted on the first reaction that is unique to purine synthesis—transfer of an amino group to PRPP to form 5-phosphoribosylamine. This reaction is catalyzed by the allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the end products IMP, AMP, and GMP. AMP and GMP act synergisti-cally in this concerted inhibition. Thus, whenever either AMP or GMP accumulates to excess, the first step in its biosynthesis from PRPP is partially inhibited. 

The next nine steps in purine nucleotide biosynthesis leading to the synthesis of IMP (whose base is hypoxanthine) are illustrated in Figure 22.7. This pathway requires four ATP molecules as an energy source. Two steps in the pathway require N10-formyltetrahydrofolate. 

Synthesis of 5 phosphoribosylamine from PRPP and glutamine is catalized by glutamine phosphoribosyl pyrophosphate amidotransferase. This enzyme is inhibited by the purine 5 -nucleotides, AMP, GMP, and IMP—the end-products of the pathway. This is the committed step in purine nucleotide biosynthesis. 

Muscular work is accompanied by the production of ammonia, the immediate source of which is adenosine 5 -phosphate (AMP).301 302 This fact led to the recognition of another substrate cycle (Chapter 11) that functions by virtue of the presence of a biosynthetic pathway and of a degradative enzyme in the same cells (cycle A, Fig. 25-17). This purine nucleotide cycle operates in the brain303 304 as well as in muscle. The key enzyme 5-AMP aminohydrolase (AMP deaminase step a, Fig. 25-17) also occurs in erythrocytes and many other tissues.304 305 Persons having normal erythrocyte levels but an absence of this enzyme in muscles suffer from muscular weakness and cramping after exercise.306... 

The biosynthesis of histidine. The 5-aminoimidazole-4-carboxamide ribotide formed during the course of histidine biosynthesis is also an intermediate in purine nucleotide biosynthesis. Therefore it can be readily regenerated to an ATP, thus replenishing the ATP consumed in the first step in the histidine biosynthetic pathway (see fig. 23.13). 

The pathway from PRPP to the first complete purine nucleotide, inosine monophosphate (IMP), involves 10 steps and is shown in figure 23.10. It would seem logical that the purine should be built up first, followed by addition of ri-bose-5-phosphate, but this is not the case. The starting point is PRPP, to which the imidazole ring is added the six-member ring is built up afterward. 

Acivicin is a potent inhibitor of several steps in purine nucleotide biosynthesis that utilize glutamine. The enzymes it inhibits are glutamine PRPP amidotransferase (step 1, fig. 23.10), phosphoribosyl-A-formylglycinamidine synthase (step 4, fig. 23.10), and GMP synthase (see fig. 23.11). In pyrimidine nucleotide biosynthesis the enzymes inhibited are carbamoyl synthase (step 1, fig. 23.13) and CTP synthase (see fig. 23.14). Acivicin is under trial for the treatment of some forms of cancer. 

Many lines of evidence indicate that the first committed step in de novo purine nucleotide biosynthesis, production of 5-phosphoribosylamine by glutamine PRPP amidotransfer-ase, is rate-limiting for the entire sequence. Consequently, regulation of this enzyme is probably the most important factor in control of purine synthesis de novo (fig. 23.24). The enzyme is inhibited by purine-5 -nucleotides, but the most inhibitory nucleotides vary with the source of the enzyme. Inhibition constants (A, ) are usually in the range 10-3-10-5 M. The maximum effect of this end-product inhibition is produced by certain combinations of nucleotides (e.g., AMP and GMP) in optimum concentrations and ratios, indicating two kinds of inhibitor binding sites. This is an example of a concerted feedback inhibition. 

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dUiydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as 5-formyl-tetrahydrofolate, leucovorin) to replete the normal tissues and avoid induction of folate deficiency- so-called leucovorin rescue. As well as depleting tissue pools of tetrahydrofolate, methotrexate leads to the accumulation of relatively large amounts of 10-formyl-dihydrofolate, which is apotentinhibitor of both thymidylate synthetase and glycinamide ribotide transformylase, an intermediate step in purine nucleotide synthesis. It is likely that this, rather than simple depletion of tetrahydrofolate, is the basis of the cytotoxic action of methotrexate (Barametal., 1988). 

Dihydrofolate Reductase. The reduced form of folate (tetrahydrofolate) acts as a one-carbon donor in a wide variety of biosynthetic transformations. This includes essential steps in the synthesis of purine nucleotides and of thymidylate, essential precursors to EHA and I A. For this reason, folate-dependent enzymes have been useful targets for the development of anticancer and anti-inflammatory drugs Ce.g., methotrexate) and anti-infectives (trimethoprim, pyrimethamine). During the reaction catalyzed by thymidylate synthase (TS), tetrahydrofolate also acts as a reducltant and is converted stoichiometrically to dihydrofolate. The regeneration of tetrahydrofolate, required for the continuous fimc-tioning of this cofactor, is catalyzed by dihydrofolate reductase (DHFR). 

The committed step in purine nucleotide biosynthesis is the conversion of PRPP into phosphoribosylamine by glutamine phosphoribosyl amidotransferase. This important enzyme is feedback-inhibited by many purine ribonucleotides. It is noteworthy that AMP and GMP, the final products of the pathway, are synergistic in inhibiting the amidotransferase. 

Pyrimidine biosynthesis in E. coli is regulated by the feedback inhibition of aspartate transcarbamoylase, the enzyme that catalyzes the committed step. CTP inhibits and ATP stimulates this enzyme. The feedback inhibition of glutamine-PRPP amidotransferase by purine nucleotides is important in regulating their biosynthesis. 

Muscle can metabolize AMP by using the purine nucleotide cycle. The initial step in this cycle, catalyzed by AMP deaminase, is the conversion of AMP into IMP. 

The detailed mechanism of myocardial protection via PC is not fully understood yet. Many pathways have been proposed and include myocardial stunning, synthesis of heat-shock proteins, involvement of G-proteins, and nitric oxide production [3-5]. The generally accepted model is that the ischemic phase leads to enhanced catabolism of purine nucleotides, resulting in a high level of adenosine. These activate PKC and a cascade of signaling steps leading to activation of MAP, MAPK and MAPKK, culminating in a marked effect on ATP-dependent channels [3,4,6, ]. 

Salvage pathways of purine nucleotide synthesis. The preformed purines can be converted to mononucleotides in a single step, using PRPP. 

Depletes the supply of PRPP needed for the first step of de novo purine nucleotide biosynthesis... 

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,... 

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