Hypoxanthine salvage transferase

An alternative mechanism of SAB action could involve its known effects on de novo purine biosynthesis (1, S) and/or nucleoside transport (5). The combined inhibitory effects of SAB and purine analogues on purine biosynthesis could result in sufficient depletion of intracellular nucleotide pools to result in enhanced cellular cytotoxicity. In addition, these effects would lead to an increased bioavailability of 5-phosphoribosyl-l-pyrophosphate (PRPP), the first enzymic product in the de novo pathway. Increased PRPP levels would enhance the activity of hypoxanthine phosphoribosyl transferase, leading to increased salvage of purine analogues. 

Some rare inherited deficiencies of the purine-salvage enzymes hypoxanthine-phosphoribosyl-transferase (HPRT) and adenine-PRT (APRT) lead to primary purine overproduction (Table 20.5). X-linked Lesch-Nyhan syndrome occurs in complete deficiency of HPRT. It is characterized by mental retardation, self-mutilation, choreoathetosis, gout. 

Two phosphoribosyl transferases then convert adenine to AMP and hypoxanthine and guanine to IMP or GMP (Figure 34-4). A second salvage mechanism involves phosphoryl transfer from ATP to a purine ri-bonucleoside (PuR) ... 

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. 

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. 

Answer D. IMP is a feedback inhibitor of PRPP amidophosphoribosyl transferase, the first reaction in the biosynthesis of purines. IMP is formed by the HPRT reaction in the salvage of hypoxanthine. 

Azathioprine [a zah THIO preen] has been the cornerstone of immunosuppressive therapy over the last several decades. It has a nitroimidazoloyl side chain attached to the sulfur of 6-mercap-topurine, which is removed by non-enzymatic reduction in the body by glutathione to yield 6-mercaptopurine (6-MP). The latter is then converted to the corresponding nucleotide, thioinosinic acid (TIMP), by the salvage pathway enzyme, hypoxanthine-gua-nine phosphoribosyl transferase. The immunosuppressant effects of azathioprine are due to this fraudulent nucleotide. (See pp. 380-381 for a discussion of 6-MP s mechanism of action, resistance, pharmacokinetics, and adverse effects.) Because of their rapid proliferation in the immune response, and their dependence on de novo synthesis of purines required for cell division, lymphocytes are predominantly affected by the cytotoxic effects of azathioprine. The drug has little effect on suppressing a secondary immune response. 

HAT medium. Mutant cells unable to synthesize nucleotides by salvage pathways are very useful tools in molecular and cell biology. Suppose that cell A lacks thymidine kinase, the enzyme catalyzing the phosphorylation of thymidine to thymidylate, and that cell B lacks hypoxanthine-guanine phosphoribosyl transferase. 

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. 

Figure 7-21. Salvage of the purine bases guanine, adenine, and hypoxanthine occurs in reactions catalyzed by phosphoribosyl transferases.

Lesch-Nyhan syndrome is caused by a defective hypoxanthine-gua-nine phosphoribosyl transferase (HGPRT). Purine bases cannot be salvaged (i.e., reconverted to nucleotides). The purines are converted to uric acid, which rises in the blood. Mental retardation and self-mutilation are characteristics of the disease. 

Fig. 5.1. Two pathways in normal cells to synthesize DNA precursors. The mutant myeloma cell line, which has the capacity to grow indeflnitely in vitro and can confer this property on antibody-producing lymphocytes through cell fusion, lacks the salvage pathway (no thymidine kinase (TK) or hypoxanthine-guanine phosphori-bosyl transferase (HGPRT)). This cell line would, therefore, not grow in the presence of folic acid antagonists. Fused cells, however, may grow since the antibody-producing cells contribute the salvage pathway.

Many cells, however, are resistant to antifolates because they contain enzymes that can synthesize the necessary nucleotides from purine bases and thymidine (Figure 6-39, bottom). Two key enzymes In these nucleotide salvage pathways are thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Cells that produce these enzymes can grow on HAT medium, which supplies a... 

Fig. 4-14). Both 6-mercaptopurine and 6-thioguanine are not active purine inhibitors until they are converted to their respective nucleotides by hypoxanthine-guanine phosphoribo-syl transferase enzymes in the cell. Since this is an intrinsically destructive process to the cell, it is referred to as a lethal synthesis or salvage pathway. Tumor cells or normal cells that have very low levels of this enzyme are resistant to these two agents. Absence of this enzymatic function is not lethal to the cell since the synthesis of purines in the cell can occur by different pathways. 

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT), a salvage enzyme of nucleotide metabolism, uses 5 -phosphoribosylpyrophosphate (PRPP) to convert hypox-... 

The base is directly ribosylated with PRPP by PT. The two enzymes concerned with purine salvage are adenine phosphoribosyl transferase (APRT), which catalyzes the formation of AMP, and hypoxanthine-guanine phosphoribosyl transferase (HGPRT), which catalyzes the formation of GMP as well as IMP, the precursor of GMP and AMP (Figure 6.58). Purine nucleotide phosphorylases (PNPases) can also... 

Once the fusion has taken place, it is necessary to eliminate any unfused myeloma cells and to select only hybrid cells secreting antibody. This is primarily achieved by the use of hypoxanthine aminopterin thymidine (HAT) media and cells that are deficient in the enzyme responsible for incorporation of hypoxanthine into DNA. Figure 3 illustrates this process. The unfused splenocytes are not immortal and naturally die off in culture. The elimination of the unfused myeloma cells is carried out by the initial use of mutant myeloma cells selected for a deficiency in the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK), rendering them unable to use the salvage pathway for nucleic acid synthesis. The myelomas will die off... 

The quantitative importance of the salvage pathway in purine metabolism is difficult to estimate [156]. Comparison between uric acid production in normal children and in children affected with a deficiency in hypoxanthine guanine phosphoribosyl transferase has, however, permitted researchers to approximate how much the salvage pathway contributes to purine metabolism. Thus, whereas in normal children uric acid excretion in the urine (per 24 hours and per kilogram of body weight) is of the order of 10 mg, in children affected with the Lesch-Nyhan syndrome, uric acid excretion is around 47 mg. The difference between enzyme-deficient and normal children is believed to reflect the amount of uric acid normally used in the salvage pathway. 

The biochemical mechanism regulating the balance between de novo and salvage pathways remains to be discovered. But observation of patients afflicted with gout or the Lesch-Nyhan syndrome suggests that hypoxanthine guanine phosphoribosyl transferase may play an important role in maintaining the balance between de novo and salvage pathways. 

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