Adenine phosphoribosyltransferase enzyme

Adenine phosphoribosyltransferase (APRT) deficiency is an inherited disorder of purine metabolism and is inherited in an autosomal recessive manner (K18, V7). This enzyme deficiency results in an inability to salvage the purine base adenine, which is oxidized via the 8-hydroxy intermediate by xanthine oxidase to 2,8-di-hydroxyadenine (2,8-DHA). This produces crystalluria and the possible formation of kidney stones due to the excretion of excessive amounts of this insoluble purine. Type I, with virtually undetectable enzyme activity, found predominantly in Caucasians, is found in homozygotes or compound heterozygotes for null alleles. Type II, with significant APRT activity, found only in Japan, is related to a missense mu-... 

The enzymatic conversion of many analogues of the naturally occurring purines directly to their biologically active form, the ribonucleotides, in vivo [5, 8, 10, 13, 39] underlines the importance of these enzymes to the drug action of this class of compounds. 2-Aminoadenine (2, 6-diaminopurine, I) [107], 2-fluoroadenine (II) [108], 4-aminopyrazolo [3, 4-d] pyrimidine (VIll) [109]. and 2- and 8-aza-adenine (IX and X) [ 110, 111] have all been shown to be substrates for the adenine phosphoribosyltransferase [J12, 113]. Extensive studies on the metabolism of 2-aminoadenine (I) in E. coli [114, 115], L cells [116], and mice [117] have also shown its conversion by this enzyme to the ribonucleotide. 

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. 

Purine salvage pathway The synthesis of purine nucleotides by the condensation of the purine bases with phosphoribosyl pyrophosphate. As the name suggests, it is a way in which purine bases can be recycled back to nucleotides. The purine salvage pathway consists of two enzymes, HGPRT and adenine phosphoribosyltransferase (APRT). 

One more enzyme belongs to this system, converting a free base directly into a ribosides -monophosphate (adenine phosphoribosyltransferase, EC 2.4.2.7). The enzyme partially purified from wheat germ [123] converted iPA into iPARMP moreover, the crude enzymes extracted from Arabidopsis thaliana and Lycopersicon esculentum plants were able to convert also BA into BARMP [124,125, respectively]. 

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.

Figure 3. Compartmentalization of the purine salvage pathway of Leishmania. Abbreviations are as follows AAH, adenine aminohydrolase XPRT, xanthine phosphoribosyltransferase HGPRT, hypoxanthine-guaninephosphoribosyltransferase ADSS, adenylosuccinate synthetase ASL, adenylosuccinate lyase IMPDH, inosine monophosphate dehydrogenase GMPS, gua-nosine monophosphate synthase GDA, guanine deaminase AMPDA, adenosine monophosphate deaminase GMPR, guanosine monophosphate reductase APRT, adenine phosphoribosyltransferase AK, adenosine kinase. Enzymes that have been localized are shown in black and those that are predicted to be in the denoted locations are depicted in gray.

No evidence for the presence of a known enzyme abnormality causing purine overproduction could be obtained. The erythrocyte activity of hypoxanthine-guanine phos-phoribosyltransferase (HGPRT), of adenine phosphoribosyltransferase (APRT), and of phosphoribosylpyrophosphate (PRPP) synthetase were all in the normal range. Erythrocyte PRPP generation, as well as the acitivity of the pentose phosphate pathway was also normal (Table 1). In addition, the rate of de novo synthesis of purine nucleotides in cultured skin fibroblasts from the patient was found to be normi. 

It cannot be ruled out that RNA transcription is unimportant for normal development prior to blastocyst formation. The fertilized egg contains many enzymes, and the activity of these has been shown to change considerably through preimplantation development. For example, the activity of hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, and hexokinase increases markedly from the fertilized egg to the blastocyst stage (Brinster, 1968a Epstein, 1970) ... 

The tissue distribution of adenine phosphoribosyltransferase varies considerably in rat, mouse, man, and monkey, and representative data are shown in Table 8-1. Murray 30) has found that the kinetic parameters of this enzyme in mouse liver vary during the course of embryonic development and has suggested that two forms of the enzyme may appear in succession. Both Murray 30) and Epstein 31) have observed marked changes in adenine phosphoribosyltransferase activity during embryonic development. 

Stadtman 65) and Blakley and Vitols 66) have reviewed studies of the inhibition and stimulation of the purine phosphoribo ltransferases by purine ribo- and deoxyribonucleotides. At relatively high concentrations, a variety of nucleotides inhibit these enzymes, while a few increase these activities at low concentrations. Studies by Henderson et al. 67) have shown that inhibitors bind to several kinetically significant forms of adenine phosphoribosyltransferase and that there are probably several different inhibitor binding sites which are not the same as those to which substrates and products bind. It must be emphasized, however, that the physiological significance of the studies conducted so far is unclear. Attempts to study the control of these reactions in intact cells have merely emphasized the complexity of this control. Under some conditions the availability of PP-ribose-P may limit the rate of these reactions. 

Adenine phosphoribosyltransferase (APRT) is a relatively non-abundant soluble enzyme which in man is coded for by a single structural gene on chromosome 16 (1). A partial deficiency of APRT in man was first described in 1968 (2). These subjects were asymptomatic and were shown to be heterozygous for the enzyme defect. 

J.M. Wilson, P.E. Daddona, H.A. Simmonds, K.J. Van Acker, and W.N. Kelley, Human adenine phosphoribosyltransferase immunochemical quantitation and protein blot analysis of mutant forms of the enzyme, Biol. Chem. 257 1508-1515 (1982). 

Increased intracellular levels of PP-ribose-P have been implicated in the cause of certain hyperuricemic states associated with uric acid overproduction. Fibroblasts from two patients with the Lesch-Nyhan syndrome were found previously to have an elevated intracellular concentration of PP-ribose-P with a normal rate of PP-ribose-P production (Rosenbloom, et al., 1968). Green and Seegmiller (1969) subsequently reported a mean PP-ribose-P value of 47.1 in erythrocytes from seven patients with HGPRT deficiency. We have confirmed these elevated PP-ribose-P levels in three additional patients with the Lesch-Nyhan syndrome with values of 20.5, 39.4 and 49.5 juM (Table 1). The mothers of these patients are obligate heterozygotes and have normal PP-ribose-P levels. Two diseases associated with a deficiency of other PRT enzymes are not associated with altered erythrocyte PP-ribose-P levels (Table 1). PP-ribose-P levels were in the normal range in one patient with a partial deficiency of adenine phosphoribosyltransferase (APRT) and in one patient with orotic aciduria, which is due to a deficiency... 

The most highly purified enzyme preparation was studied for substrate specificity (Table 2). The results are expressed as per cent of nucleotide formed from each substrate as compared to adenine. Adenine phosphoribosyltransferase shows substantial activity toward 4-amino-5-imidazolecarboxamide and 2,6-diaminopurine but not hypoxanthine, guanine, 6-mercaptopurine, or adenosine. 

Fig ire 2 shows the possible interconversions between purine bases, nucleosides and nucleoside monophosphates. Prom genetic and enzymatic studies it has been shown that the conversion of the free bases(adenine, hypoxanthine,xanthine and guanine) to the monophosphate level is carried out by at least three different enzymes (the phosphoribosyltransferases)(1,5). This is in contrast to htunan tissues, which have one specific adenine phosphoribosyltransferase and one transferase with activity towards hypoxanthine,xanthine and guanine. 

Partial deficiency of HGPRT, a salvage enzyme of purine metabolism, has been demonstrated to be the primary abnormality causing purine overproduction in a small proportion of patients with gout (l-4). The quantitative deviation in the activity of this enzyme has been shown by Kelley et al. to be associated with decreased stability to thermal inactivation (2). These authors suggest that in the affected subjects HGPRT is structurally altered. Furthermore, in some of these patients erythrocyte adenine phosphoribosyltransferase (APRT) activity was found to be increased and relatively thermostable (2). 

The Lesch-Nyhan Syndrome (LNS) is a rare x-linked neurological disease of children characterized by choreoathetosis, spasticity, mental retardation and compulsive self mutilation accompanied by excessive purine production and hyperuricemia (l). The virtually complete deficiency of activity of a purine salvage enzyme, hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8.) (2), due to structural gene mutation (3 4) has been shown to be the basic abnormality in this disease. In erythrocytes of LNS patients, HGPRT deficiency has been found to be associated with increased activity and relative thermal stability of adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7 ) (5 6) an autosomally determined enzyme (7) ... 

Adenine phosphoribosyltransferase activity assayed by an analogous method using 0.6 mM adenine-8-l C (specific activity 4 mCI/mmole) was not significantly altered in any of the HGPRT-deficient clones (Table II). Starch gel electrophoresis using a method only slightly modified from that of Watson et. al. (19) showed no difference in electrophoretic mobility of mutants partially deficient in HGPRT (Fig. 3) but could not be used to examine the clones with severely deficient enzyme activity. 

---