UNIPROT:P00492 - FACTA Search

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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lesch-Nyhan syndrome is a hereditary disorder of purine metabolism causing overproduction of uric acid and neurological problems including spasticity, choreoathetosis, mental retardation, and compulsive self-mutilation. The syndrome is caused by a defect in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), which converts guanine and hypoxanthine to the nucleotides GMP and IMP. There is evidence that the neurological problems are due to an adverse effect of the HPRT deficiency on the survival and/or development of dopaminergic neurons, specifically. Here we report that HPRT-deficient PC12 mutants that have a normal or near normal dopamine content (55-97% of that of wild-type cells) fail to undergo neuronal differentiation induced by nerve growth factor (NGF) when the de novo pathway of purine synthesis is partially inhibited. However, nerve growth factor-induced differentiation is near normal under these conditions in PC12 HPRT-deficient mutants containing much lower dopamine levels (<8% of that of wild type cells), indicating a neurotoxic effect of the endogenous dopamine in the mutants. The degree of inhibition of the de novo pathway of purine synthesis was the same in both classes of HPRT-deficient mutants. Expression of BCl-2 in a PC12 mutant that has a normal dopamine content allowed partial NGF-induced differentiation suggesting that the apoptotic pathway might be involved in the failure of differentiation when the de novo pathway of purine synthesis is partially inhibited.
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PMID:Impaired differentiation of HPRT-deficient dopaminergic neurons: a possible mechanism underlying neuronal dysfunction in Lesch-Nyhan syndrome. 967 Sep 94

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been proven to be a target for potential anti-tritrichomonial chemotherapy. Using a structure-based approach, the base-binding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determining the roles of different active-site residues in purine specificity would form the basis for designing specific inhibitors toward the parasitic enzyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longer recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recognize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the Kms for GMP and XMP (but not IMP). This change leads to increased kcats for the forward reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr156, resulted from mutations of Arg155, bring little effect on the hydrophobic interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which involve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain carboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 was proven to be the primary determinant in conferring the specificity of the enzyme toward 6-oxopurines. By substituting the lysine residue for a serine, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xanthine as its substrates. Adenine and hypoxanthine are equivalent substrates for the mutant enzyme with similar Kms of 34.6 and 38.0 microM, respectively. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AMP with an estimated Kis of 25.4 microM against alpha-D-5-phosphoribosyl-pyrophosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetusHGXPRTase into either a HGPRTase or a genuine AHGXPRTase.
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PMID:Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein. 984 28

Guanine phosphoribosyltransferase (GPRTase) from Giardia lamblia, an enzyme required for guanine salvage and necessary for the survival of this parasitic protozoan, has been kinetically characterized. Phosphoribosyltransfer proceeds through an ordered sequential mechanism common to many related purine phosphoribosyltransferases (PRTases) with alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) binding to the enzyme first and guanosine monophosphate (GMP) dissociating last. The enzyme is a highly unique purine PRTase, recognizing only guanine as its purine substrate (K(m) = 16.4 microM) but not hypoxanthine (K(m) > 200 microM) nor xanthine (no reaction). It also catalyzes both the forward (kcat = 76.7 s-1) and reverse (kcat = 5.8.s-1) reactions at significantly higher rates than all the other purine PRTases described to date. However, the relative catalytic efficiencies favor the forward reaction, which can be attributed to an unusually high K(m) for pyrophosphate (PPi) (323.9 microM) in the reverse reaction, comparable only with the high K(m) for PPi (165.5 microM) in Tritrichomonas foetus HGXPRTase-catalyzed reverse reaction. As the latter case was due to the substitution of threonine for a highly conserved lysine residue in the PPi-binding loop [Munagala et al. (1998) Biochemistry 37, 4045-4051], we identified a corresponding threonine residue in G. lamblia GPRTase at position 70 by sequence alignment, and then generated a T70K mutant of the enzyme. The mutant displays a 6.7-fold lower K(m) for PPi with a twofold increase in the K(m) for PRPP. Further attempts to improve PPi binding led to the construction of a T70K/A72G double mutant, which displays an even lower K(m) of 7.9 microM for PPi. However, mutations of the nearby Gly71 to Glu, Arg, or Ala completely inactivate the GPRTase, suggesting the requirement of flexibility in the putative PPi-binding loop for enzyme catalysis, which is apparently maintained by the glycine residue. We have thus tentatively identified the PPi-binding loop in G. lamblia GPRTase, and attributed the relatively higher catalytic efficiency in the forward reaction to the unusual loop structure for poor PPi binding in the reverse reaction.
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PMID:Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis. 1009 38

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.
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PMID:Consequences of IMP dehydrogenase inhibition, and its relationship to cancer and apoptosis. 1039 Jun 1

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the phosphoribosylation of hypoxanthine and guanine by transferring the phosphoribosyl moiety from phosphoribosylpyrophosphate (PRPP) on to N9 in the purine base, resulting in the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP). Xanthine is an additional substrate for the Plasmodium falciparum HGXPRT. Our aim has been to elucidate structural features in HGPRT that govern substrate specificity. We have addressed this problem by engineering chimeric HGPRTs, which contain segments from both the parasite and human enzymes. Four chimeric enzymes were engineered (DS1-DS4), of which the chimeric enzyme, DS1, in which the first 49 residues of human HGPRT were replaced with the corresponding residues from the P. falciparum enzyme, exhibited additional specificity for xanthine. None of the switched residues forms a part of the purine or PRPP binding region in the available crystal structures of HG(X)PRTs. Our data on the chimeric enzyme DS1 provide the first evidence that the N-terminal approximately 50 amino acids, although not proximal to the active site in the crystal structure, can in fact modulate substrate specificity. DS1 exhibits a reduced k(cat) for hypoxanthine and guanine, while its K(m) for these oxopurine bases remains largely unchanged. Its specific activity for xanthine is comparable with hypoxanthine but five times more than that for guanine.
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PMID:Unusual substrate specificity of a chimeric hypoxanthine-guanine phosphoribosyltransferase containing segments from the Plasmodium falciparum and human enzymes. 1083 58

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8.; HPRT) catalyzes the salvage synthesis of inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) from the purine bases hypoxanthine and guanine, respectively. Complete deficiency of HPRT activity is associated with the Lesch-Nyhan syndrome (LNS), characterized by excessive purine production and severe neurological manifestations. The etiology of the metabolic consequences of HPRT deficiency is clarified, but that of the neurological manifestations is not yet understood. HPRT-deficient mice represent an experimental animal model of LNS. In search for a possible metabolic abnormality in LNS brains, connecting the neurological deficit to HPRT deficiency, the purine and pyrimidine nucleotide content of cultured neurons, prepared from HPRT-deficient transgenic mice, was now determined. The HPRT-deficient neuronal cultures exhibited a significantly elevated content of the pyrimidine nucleotides UTP (1.33-fold the normal level, p = 0.0002) and CTP (1.28-fold the normal level, p = 0.02), but normal content of the purine nucleotides ATP and GTP. This abnormality in neuronal pyrimidine nucleotide content may be associated with the pathophysiology of the neurological deficit in LNS.
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PMID:Elevated UTP and CTP content in cultured neurons from HPRT-deficient transgenic mice. 1085 40

Lesch-Nyhan syndrome is a metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch-Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5'-nucleotidase activity in the erythrocytes of seven patients with Lesch-Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5'-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch-Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.
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PMID:Cytosolic 5'-nucleotidase hyperactivity in erythrocytes of Lesch-Nyhan syndrome patients. 1088 27

Guanine phosphoribosyltransferase from Giardia lamblia, a key enzyme in the purine salvage pathway, is a potential target for anti-giardiasis chemotherapy. Recent structural determination of GPRTase (Shi, W., Munagala, N. R., Wang, C. C., Li, C. M., Tyler, P. C., Furneaux, R. H., Grubmeyer, C., Schramm, V. L., and Almo, S. C. (2000) Biochemistry 39, 6781-6790) showed distinctive features, which could be responsible for its singular guanine specificity. Through characterizing specifically designed site-specific mutants of GPRTase, we identified essential moieties in the active site for substrate binding. Mutating the unusual Tyr-127 of GPRTase to the highly conserved Ile results in 6-fold lower K(m) for guanine. A L186F mutation in GPRTase increased the affinity toward guanine by 3. 3-fold, whereas the corresponding human HGPRTase mutant L192F showed a 33-fold increase in K(m) for guanine. A double mutant (Y127I/K152R) of GPRTase retained the improved binding of guanine and also enabled the enzyme to utilize hypoxanthine as a substrate with a K(m) of 54 +/- 15.5 microm. A triple mutant (Y127I/K152R/L186F) resulted in further increased binding affinity with both guanine and hypoxanthine with the latter showing a lowered K(m) of 29.8 +/- 4.1 microm. Dissociation constants measured by fluorescence quenching showed 6-fold tighter binding of GMP with the triple mutant compared with wild type. Thus, by increasing the binding affinity of 6-oxopurine, we were able to convert the GPRTase to a HGPRTase.
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PMID:Converting the guanine phosphoribosyltransferase from Giardia lamblia to a hypoxanthine-guanine phosphoribosyltransferase. 1097 10

We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.
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PMID:Yeast GMP kinase mutants constitutively express AMP biosynthesis genes by phenocopying a hypoxanthine-guanine phosphoribosyltransferase defect. 1106 76

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the reutilization of hypoxanthine and guanine to the purine nucleotides IMP and GMP, respectively. HPRT deficiency is an X-linked disorder characterized by uric acid overproduction and variable neurologic impairment. The complete deficiency of HPRT is diagnostic of Lesch-Nyhan syndrome manifested by choreoathetosis, spasticity, mental retardation, and self-injurious behavior. In some HPRT-deficient patients the enzyme defect appeared to be "partial" and the neurologic symptoms mild to severe (Kelley-Seegmiller syndrome). This has prompted the classification of HPRT deficiency in 2 distinct groups: Lesch-Nyhan syndrome and Kelley-Seegmiller syndrome, which has created much confusion. A spectrum of clinical consequences of HPRT deficiency has been recognized in small series of patients, but the complete spectrum of the neurologic disorder has not been described in a single series of patients examined by the same observers. We analyzed our experience with 22 patients belonging to 18 different families with HPRT deficiency diagnosed at "La Paz" University Hospital in Madrid over the past 16 years. The clinical spectrum of these HPRT-deficient Spanish patients was similar to the different phenotypes occasionally reported in the literature, in some cases diagnosed as Lesch-Nyhan "variants." The clinical, biochemical, enzymatic, and molecular genetic studies on these 22 patients allowed us to delineate a new classification of HPRT deficiency. Based on the neurologic symptoms, dependency for personal care, HPRT activity in hemolysate and in intact erythrocytes, and predicted protein size, patients were classified into 4 groups: Group 1 (2 patients), normal development with no neurologic symptoms, HPRT activity was detectable in hemolysates and in intact erythrocytes, and the mutation did not affect the predicted protein size. Group 2 (3 patients) mild neurologic symptoms that did not prevent independent lives, HPRT activity was detectable in intact erythrocytes, and the protein size was normal. Group 3 (2 patients), severe neurologic impairment that precluded an independent life, no residual HPRT activity, and normal protein size. Group 4 (15 patients), clinical characteristics of Lesch-Nyhan syndrome (some may not show self-injurious behavior), no residual HPRT activity, and in most (7 of 8 patients in whom the mutation could be detected) the mutation affected the predicted protein size. This classification of HPRT deficiency into 4 groups may be more useful in terms of accuracy, reproducibility, assessment for treatment trials and prognosis. The study of this Spanish series allows us to conclude that HPRT deficiency may be manifested by a wide spectrum of neurologic symptoms; the overall severity of the disease is associated with mutations permitting some degree of residual enzyme activity; and mutation analysis provides a valuable tool for prognosis, carrier identification, and prenatal diagnosis.
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PMID:The spectrum of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency. Clinical experience based on 22 patients from 18 Spanish families. 1130 86

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