Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In cancer cells, particularly in leukaemic cells, guanylate biosynthesis is up-regulated as shown by the increased activities of IMP dehydrogenase, the rate-limiting enzyme of de novo GTP biosynthesis, and of the salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). In enzyme pattern-targeted chemotherapy, tiazofurin inhibits IMP dehydrogenase activity in cancer cells and allopurinol-induced high serum hypoxanthine levels inhibit HGPRT activity. A triad of responses was observed in the blast cells of patients treated with tiazofurin infusions: chemotherapy, induced differentiation, and down-regulation of c-Ki-ras and c-myc oncogenes. Tiazofurin was synergistic in cytotoxicity and in causing differentiation with ribavirin, retinoic acid, and gemcitabine [corrected]. Induced differentiation plays an important role in the overall impact of antipurine agents.
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PMID:Role of differentiation induction in action of purine antimetabolites. 803 45

The crystal structure of HGPRTase with bound GMP has been determined and refined to 2.5 A resolution. The enzyme has a core alpha/beta structure resembling the nucleotide-binding fold of dehydrogenases, and a second lobe composed of residues from the amino and carboxy termini. The GMP molecule binds in an anti conformation in a solvent-exposed cleft of the enzyme. Lys-165, which forms a hydrogen bond to O6 of GMP, appears to be critical for determining the specificity for guanine and hypoxanthine over adenine. The location of active site residues also provides evidence for a possible mechanism for general base-assisted HGPRTase catalysis. A rationalization of the effects on stability and activity of naturally occurring single amino acid mutations of HGPRTase is presented, including a discussion of several mutations at the active site that lead to Lesch-Nyhan syndrome.
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PMID:The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP. 804 44

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.
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PMID:Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme? 815 32

The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been determined and refined against X-ray data to 1.9 A resolution. T. foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.
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PMID:Crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus. 867 28

We found that when human promyelocytic leukemic cells (HL-60 cells) were induced to differentiate along the granulocytic lineage by two diverse mechanisms (starvation for an essential amino acid or treatment with DMSO), there was a marked decrease in the intracellular guanosine 5'-triphosphate (GTP) concentration with no change in the guanosine 5'-diphosphate (GDP) concentration. Differentiation was prevented by guanine or guanosine in a dose-dependent manner. We showed that: (a) guanine had to be converted to a nucleotide because it did not prevent differentiation of hypoxanthine-guanine phosphoribosyltransferase-deficient HL-60 cells; (b) the effect of guanine correlated with a return of the cytosolic GTP:GDP ratio to normal; and (c) other purine bases were not effective. We hypothesized that the decreased GTP:GDP ratio in differentiating HL-60 cells might decrease the relative amount of GTP bound to Ras, a key regulatory GTP-binding protein important to cell growth and differentiation. Consistent with data showing that HL-60 cells harbor an activating N-Ras mutation, we found that the percentage of Ras molecules in the GTP-bound state was high in proliferating HL-60 cells (27 +/- 3%) compared with other cultured mammalian cells (< 1%); however, we found no change in the activation state of Ras when cells ceased to proliferate and differentiated in response to DMSO, amino acid deprivation, or inhibitors of guanylate synthesis. We conclude that: (a) a decrease in the intracellular GTP concentration is necessary for HL-60 cells to undergo granulocytic differentiation; and (b) although a high degree of Ras activation contributes to the malignant phenotype of the cell, there is no change in the activation state of Ras during granulocytic differentiation.
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PMID:A decrease in the intracellular guanosine 5'-triphosphate concentration is necessary for granulocytic differentiation of HL-60 cells, but growth cessation and differentiation are not associated with a change in the activation state of Ras, the transforming principle of HL-60 cells. 899 34

The arginine-specific reagents phenylglyoxal and butane-2,3-dione irreversibly inactivate the Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) and Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The inactivation of the tritrichomonal enzyme by phenylglyoxal follows time-dependent and concentration-dependent pseudo-first-order kinetics. Complete protection against inactivation is afforded by the addition of 25 microM GMP, whereas 5-phosphoribosyl-1-diphosphate (PRibPP) at 50-250 microM can only slow down the inactivation, without being protective. Digestion of [7-(14)C]phenylglyoxal-modified enzyme with trypsin and separation of the peptides by reverse-phase HPLC shows that only one radioactive peak is greatly diminished by incubation with 25 microM GMP or 1 mM PRibPP. Mass-spectral analysis identifies Arg155 as the target site of two molecules of phenylglyoxal that is protected by the substrates. This amino acid residue is positioned next to Tyr156, which is a highly conserved aromatic residue among all the purine phosphoribosyltransferases (PRT) and is always found stacked on top of the purine substrate. This may explain why phenylglyoxal labeling of Arg155 inactivates the enzyme and why GMP can protect Arg155 more effectively than PRibPP. Among the purine PRT in our possession, only schistosomal HGPRT, the only other enzyme that contains an arginine residue at the corresponding location (Arg187), was susceptible to phenylglyoxal and butane-2,3-dione. The presence of Lys185-Phe186 and Ser179-Trp180 at the corresponding locations in human HGPRT and Giardia lamblia GPRT, respectively, may explain their resistance to phenylglyoxal. Thus, Arg155 in T. foetus HGXPRT and Arg187 in S. mansoni HGPRT will be attractive targets for future studies.
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PMID:Inactivation of Tritrichomonas foetus and Schistosoma mansoni purine phosphoribosyltransferases by arginine-specific reagents. 910 51

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of Lesch-Nyhan syndrome, the activator of the prodrugs 6-mercaptopurine and allopurinol, and a target for antiparasitic chemotherapy. The three-dimensional structure of the recombinant human enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here, ligand binding, pre-steady state kinetics, isotope trapping, and isotope exchange experiments are presented which detail the sequential kinetic mechanism of the enzyme. In the forward reaction, in which a base (hypoxanthine or guanine) reacts with PRPP to form nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction, IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of guanine than hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product guanine to stimulate, rather than inhibit, the pyrophosphorolysis of IMP. The overall equilibrium for the hypoxanthine phosphoribosyl transfer reaction lies far toward nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked nucleotide formation. The three-dimensional structure of the HGPRTase x IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the ligand binding and kinetic studies are discussed in light of the structural data.
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PMID:Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry. 913 23

Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi-bi mechanism, where the substrates bind to the enzyme (first PRPP followed by the purine bases), and the products released (first PPi followed by purine nucleotide) in a defined order. The Kms for PPi in the T. foetus HGXPRTase-catalyzed reactions are unusually high, close to the millimolar range. Since the crystal structure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-7040] suggests potential binding between the threonine-47 in a conserved cis-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al. (1994) Cell 78, 325-334] at the corresponding position, we prepared a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10-fold decrease of Km for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the active site as compared to the human HGPRTase are thus proposed to be responsible for the high Km for PPi in the T. foetus HGXPRTase-catalyzed reaction.
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PMID:Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. 952 25

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes the reversible formation of IMP and GMP from their respective bases hypoxanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phosphoribosyl-1-pyrophosphate (PRPP). The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine moiety, allowing enzymic catalysis to reduce the energy barrier associated with the reaction. The pH profile of kcat for IMP pyrophosphorolysis revealed an essential acidic group with pKa of 7.9 whereas those for IMP or GMP formation indicated involvement of essential basic groups. Based on the crystal structure of human HGPRTase, protonation/deprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have constructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis. D137N displayed an 18-fold decrease in kcat for nucleotide formation with Hx as substrate, a 275-fold decrease in kcat with Gua, and a 500-fold decrease in kcat for IMP pyrophosphorolysis. D137N also showed lower KD values for nucleotides and PRPP. The pH profiles of kcat for D137N were severely altered. In contrast to D137N, the kcat for K165Q was decreased only 2-fold in the forward reaction and was slightly increased in the reverse reaction. The Km and KD values showed that K165Q interacts with substrates more weakly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type. In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (3000-fold reduction) was affected much more than hypoxanthine (32-fold reduction). In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrates.
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PMID:Catalysis in human hypoxanthine-guanine phosphoribosyltransferase: Asp 137 acts as a general acid/base. 952 33

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.
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PMID:Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan. 954 15


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