Purine salvages hypoxanthine

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

Lesch-Nyhan syndrome, an overproduction hyperuricemia characterized by frequent episodes of uric acid hthiasis and a bizarre syndrome of self-mutilation, reflects a defect in hypoxanthme-guanine phosphoribo-syl transferase, an enzyme of purine salvage (Figure 34—4). The accompanying rise in intracellular PRPP results in purine overproduction. Mutations that decrease or abohsh hypoxanthine-guanine phosphoribosyltrans-ferase activity include deletions, frameshift mutations, base substitutions, and aberrant mRNA splicing. 

This enzyme [EC 2.4.2.8] (also known as hypoxanthine phosphoribosyltransferase, IMP pyrophosphorylase, transphosphoribosidase, and guanine phosphoribosyltransferase) catalyzes the purine salvage reaction of hypoxanthine with 5-phospho-a-D-ribose 1-diphosphate to... 

The role of hypoxanthine-guanine phosphoribosyltransferase in purine salvage has been confirmed by the abnormally high excretion of purines (as uric acid) in humans who lack hypoxanthine-guanine phosphoribosyltransferase. Studies of purine metabolism in cultures of cells from patients with this hereditary disorder also support this conclusion. 

Adenine phosphoribosyltransferase catalyzes the conversion of adenine to AMP in many tissues, by a reaction similar to that of hypoxanthine-guanine phosphoribosyltransferase, but is quite distinct from the latter. It plays a minor role in purine salvage since adenine is not a significant product of purine nucleotide catabolism (see below). The function of this enzyme seems to be to scavenge small amounts of adenine that are produced during intestinal digestion of nucleic acids or in the metabolism of 5 -deoxy-5 -methylthioadenosine, a product of polyamine synthesis. 

The most recent application of RPLC to the analysis of enzymes has been reported by Halfpenny and Brown (HI). An assay for purine nucleoside phosphorylase, a key mediator in the purine salvage pathway, has been developed and optimal conditions for the analysis determined. Figure 20 illustrates the simultaneous separation of the substrate, inosine, and products, uric acid and hypoxanthine. In another analysis. Halfpenny and Brown (H2) developed an assay for hypoxanthine-guanine phos-phoribosyltransferase. Deficiency of this enzyme has been associated with Lesch-Nyhan syndrome as well as primary gout. The activity of the enzyme is determined by measurement of the decrease of the substrate, hypoxanthine, and increase in the product, inosine-5 -monophosphoric acid. A major advantage of using HPLC for enzyme assays is that the simultaneous measurement of both substrate and product reduces the error due to interference from competing enzymes. 

Most of the free purines derived from the breakdown of DNA, RNA, and nucleotides in the diet are catabolized to xanthine and then to uric acid in the gut mucosa. The AMP and GMP biosynthesized in the body can also be bmken down to free purines, such as adenine, guanine, and hypoxanthine. These purines, in contrast to those derived frcim the diet, are largely reused for the synthesis of ATP and GTP- They are first converted back to AMP or GMP in a pathway of reutiliza-lion called the purine salvage pathway. For example, adenine phosphoribosyl-transferase (PRPP) catalyzes the conversion of adenine to AMP. Here, PRPP serves as the source of the phosphoribose group. Pyrophosphate is a product of the reaction. 

HGPRT Hypoxanthine-guanine phosphoribosyltransferase the enzyme that catalyzes the synthesis of inosine monophosphate (IMP) and guano-sine monophosphate (GMP) from hypoxanthine and guanine, respectively. It makes up part of the purine salvage pathway, a way of recycling purine bases back to the nucleotides. 

Answer C. The purine antimetabolite 6-mercaptopurine is bioactivated in cancer cells by the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT). The most common form of resistance to 6-MP is a decrease in activity of this enzyme. Azathioprine, a drug used as an immunosuppressant, is closely related to 6-MP and also requires bioactivation to exert cytotoxic actions. 

In the purine salvage pathway, purine bases obtained from the normal turnover of cellular nucleic acids or (to a lesser extent) from the diet are reconverted into nucleotides. Because the de novo synthesis of nucleotides is metabolically expensive (i.e., relatively large amounts of phosphoryl bond energy are used), many cells have mechanisms to retrieve purine bases. Hypoxanthine-guaninephos-phoribosyltransferase (HGPRT) catalyzes nucleotide synthesis using PRPP and either hypoxanthine or guanine. The hydrolysis of pyrophosphate makes these reactions irreversible. 

HAT Medium - A cell culture medium augmented by hypoxanthine, aminopterin, and thymidine, which selects for cells that have a functional purine salvage pathway. 

HAT Selection - The compounds hypoxanthine, aminopterin (see here), and thymidine (H,A, and T, respectively) can be used to select for cells having functional salvage pathways. Aminopterin inhibits dihydrofolate reductase, which blocks de novo purine and thymidine synthesis. Only cells which can utilize thymidine (pyrimidine salvage) and hypoxanthine (purine salvage) can grow in this medium. 

The HGXPRTase activity appears to be the major route of purine salvage. Adenine is salvaged by deamination to hypoxanthine whereas adenosine can be either phos-phorylated directly or deaminated. Guanosine is directly phosphorylated to GMP or cleaved to guanine. 

Individual enzymes of purine salvage are similar to those of Leishmania. PRTase activities were found for adenine, hypoxanthine, and guanine in the three forms (43). As in Leishmania, there is also a separate xanthine PRTase. Nucleoside kinase activities were found for adenosine, inosine, and guanosine (43), nucleoside hydrolase activities for inosine and guanosine and a nucleoside phosphorylase activity for adenosine. There are both nucleoside hydrolase and phosphorylase activities in epimastigotes (44,45). The adenylosuccinate synthetase and adenylosuccinate lyase are essentially identical to those found in L. donovani (46). 

Fig. 41.10. Salvage of bases. The purine bases hypoxanthine and gnanine react with PRPP to form the nucleotides inosine and gnanosine monophosphate, respectively. The enzyme that catalyzes the reaction is hypoxanthine-gnanine phosphoribosyltransferase (HGPRT). Adenine forms AMP in a reaction catalyzed by adenine phosphoribosyltransferase (APRT). Nucleotides are converted to nucleosides by 5 -nucleotidase. Free bases are generated from nncleosides by purine nucleoside phosphorylase. Deamination of the base adenine occurs with AMP and adenosine deaminase. Of the purines, only adenosine can be directly phosphorylated back to a nucleotide, by adenosine kinase.

Hyperuricemia in Lotta Topa ne s case arose as a consequence of over-j production of uric acid. Treatment with allopurinol not only inhibits xan-thine oxidase, lowering the formation of uric acid with an increase in the excretion of hypoxanthine and xanthine, but also decreases the overall synthesis of purine nucleotides. Hypoxanthine and xanthine produced by purine degradation are salvaged (i.e., converted to nucleotides) by a process that requires the consumption of PRPP. PRPP is a substrate for the glutamine phosphoribosyl amidotransferase reaction that initiates purine biosynthesis. Because the normal cellular levels of PRPP and glutamine are below the of the enzyme, changes in the level of either substrate can accelerate or reduce the rate of the reaction. Therefore, decreased levels of PRPP cause decreased synthesis of purine nucleotides. 

FIGURE 23.24 Purine salvage, (a) Adenine is the purine in this example. There are analogous reactions for salvage of guanine and hypoxanthine (see page 697). (b) The formation of phosphoribosylpyrophosphate (PRPP). 

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