Phosphoribosylamine synthesis

Figure 20.10 The positions in the pathway for de novo purine nucleotide synthesis where GLUCOSE provides the ribose molecule and GLUTAMINE provides nitrogen atoms. The pathway begins with glucose which provides ribose 5-phosphate, via the pentose phosphate pathway (Chapter 6). Glutamine provides its amide nitrogen in two reactions formation of 5-phosphoribosylamine and formation of guanosine monophosphate (GMP) from xantho-sine 5-phosphate (XMP).

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]. 

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. 

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. 

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. 

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. 

In the next step, which is the first step uniquely related to purine synthesis, the amide nitrogen from glutamine is added to the PRPP to form 5-phosphoribosylamine, catalyzed by PRPP amidotransferase. This step can be inhibited by azaserine, an antimetabolite of glutamine. Glycine is then added, forming an amide bond. This re-... 

The control of purine synthesis is with an early stepformation of phosphoribosylamine by PRPP amidotransferase. This enzyme is partially inhibited by either AMP or GMP and strongly inhibited by AMP and GMP together. 

Both AMP and GMP inhibited purine synthesis at the level of formation of phosphoribosylamine irrespective of whether glutamine or ammonia was the N-donor. Detailed analysis of the AMP studies however was difficult because of the rapid enzymatic deamination of AMP with this enzyme preparation in the absence of GTP. 

A computer model has been constructed simulating the kinetics of the reaction sequence of Fig. 7. The isotopic enrichment of glycine is represented by the equation for the polyexponential die-away curve of hippurate shown in Fig. 3. That of the amide-N of glutamine is represented by a first order product curve derived from the enrichment of phenylacetylglutamine, which starts at zero, reaches a maximum at 3 hours, and thereafter closely approximates the die-away curve of Fig. 4. The model also includes an arbitrary convention which accelerates a late reaction of the sequence (IMP -> hypoxanthine) as a higher power function of [PP-ribose-P], in order to achieve a ratio of >1 in the expression (increase in rate of synthesis of 3-phosphoribosylamine)/(increase... 

The synthesis of purines de novo was initiated by the formation of 5-phosphoribosylamine by reaction of glutamine with 6-phosphoribosylpyro-phosphate. The latter compound was the product of reaction of ribose 5-... 

In detail, the synthesis as studied by Buchanan and Greenberg takes the following route 5-phosphoribosylamine (stemming from phosphoribosyl pyrophosphate and glutamine, as mentioned under pyrimidines) condenses with glycine to form the amide with the aid of ATP the... 

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). 

Purine biosynthesis was studied in intact cells maintained in culture and in cell free preparations In cell-free preparations the regulation of phosphoribosylamine (PRA) synthesis was studied, since this step is believed to be the rate-limiting step in novo purine biosynthesis. PRA can be synthesized according to the following reactions ... 

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