Phosphoribosylamine inhibition

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. 

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. 

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. 

Its action is very complex, and a little of it, after bio-transformation to thioguanidylic acid, is incorporated into cellular DNA (Tidd and Paterson, 1974 Parks et al., 1975). However, it is not established that this is a therapeutically important reaction. Much of it is converted, in the cell, into 6-thioinosine 5 -phosphate (TIP) (Brockman, 1963). TIP inhibits conversion of inosine 5 -phosphate to adenosine 5 -phosphate, thus bringing neogenesis of purines to a halt (Salser and Balis, 1965). It also exerts feedback inhibition of the biosynthesis of phosphoribosylamine, a carbohydrate involved in the earliest steps in purine biosynthesis (Bennett etaL, 1963). 

The reaction that catalyzes the conversion of ribosyl pyrophosphate to 5 -phosphoribosylamine is likely to be the rate-limiting step in purine biosynthesis. Of course, it is difficult to pinpoint a rate-limiting step in an intact mammal, but in vitro experiments have established a feedback inhibition of glutamine phosphoribosyl pyrophosphate amino transferase by adenylic and guanylic nucleotides (ATP, ADP, GMP, GDP, and IMP). 

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. 

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