A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity

Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K(m) and k(cat) for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes.     

Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogs as potential antimalarial agents

6-Oxopurine acyclic nucleoside phosphonates (ANPs) have been shown to be potent inhibitors of hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme of the purine salvage pathway in human malarial parasites. These compounds also exhibit antimalarial activity against parasites grown in culture. Here, a new series of ANPs, hypoxanthine and guanine 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives with different chemical substitutions in the 2'-position of the aliphatic chain were prepared and tested as inhibitors of Plasmodium falciparum (Pf) HGXPRT, Plasmodium vivax (Pv) HGPRT and human HGPRT. The attachment of an hydroxyl group to this position and the movement of the oxygen by one atom distal from N(9) in the purine ring compared with 2-(phosphonoethoxy)ethyl hypoxanthine (PEEHx) and 2-(phosphonoethoxy)ethyl guanine (PEEG) changes the affinity and selectivity for human HGPRT, PfHGXPRT and PvHGPRT. This is attributed to the differences in the three-dimensional structure of these inhibitors which affects their mode of binding. A novel observation is that these molecules are not always strictly competitive with 5-phospho-α-d-ribosyl-1-pyrophosphate. 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]hypoxanthine (iso-HPMP-Hx) is a very weak inhibitor of human HGPRT but remains a good inhibitor of both the parasite enzymes with K(i) values of 2μM and 5μM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K(i) values for the parasite enzymes by sixfold. This suggests that the covalent attachment of a second group to the ANPs mimicking pyrophosphate and occupying its binding pocket could increase the affinity for these enzymes.     

Purine metabolism in high- and low-uric acid lines of chickens: hypoxanthine/guanine phosphoribosyltransferase activities

The activity of hypoxanthine/guanine phosphoribosyltransferase (HGPRT) was examined in the livers and kidneys of two genetic lines of chickens selected for different plasma uric acid levels. Previous work demonstrated that the high-uric acid line (HUA) had significantly greater de novo uric acid synthesis rates in kidney tissue compared to the low-uric acid line (LUA). In addition, phosphoribosylpyrophosphate (PRPP) synthetase and xanthine dehydrogenase activities in livers and kidneys were significantly higher in the HUA compared to the LUA line. PRPP pool sizes were also significantly higher in both livers and kidneys of HUA birds. HGPRT activities in livers of HUA birds were significantly (P less than 0.05) greater than in LUA birds. The mean value of liver HGPRT was 7.36 +/- 0.25 pmole inosine-5'-monophosphate (IMP) and 6.05 +/- 0.27 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA lines. There were no significant differences (P greater than 0.05) in kidney HGPRT activities between the two groups. The mean value of kidney HGPRT was 52.87 +/- 1.62 pmole IMP and 50.72 +/- 1.62 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA line. Elevated liver HGPRT may serve to enhance the regeneration of PRPP in the HUA liver. Elevated liver PRPP synthetase and PRPP pool size suggest an increased flux through the de novo purine biosynthetic pathway in HUA birds. The resulting additional pyrophosphate from the glutamine PRPP amidotransferase reaction would stimulate recovery of PRPP and spare the system from a substantial loss of energy.     

A point mutation at the subunit interface of hypoxanthine-guanine-xanthine phosphoribosyltransferase impairs activity: role of oligomerization in catalysis

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from Plasmodium falciparum catalyzes the phosphoribosylation of hypoxanthine, guanine and xanthine. The functionally active form of HGXPRT is a tetramer but interface residues do not contribute to catalysis. Here we report the characterization of an interface mutant Y96C, which has a decreased k(cat), an increase in the K(m) for phosphoribosyl pyrophosphate (PRPP) and no change in K(m) for the purine bases when compared to the wild type enzyme. The mutant enzyme does not tetramerize in the presence of PRPP, unlike the wild type in which the tetramer is stabilized by PRPP. This is the first report of a HGXPRT mutation, at a unique interface where non-adjacent subunits interact, that impairs catalysis.     

Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase. Stability studies on the product-activated enzyme

Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.     

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.     

The regulation of hypoxanthine guanine phosphoribosyl transferase activity through transfer of PRPP by metabolic cooperation

We have developed a method of relating changes in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) activity to the rate of phosphoribosyl pyrophosphate (PRPP) synthesis in isolated cell lines and in co-cultures of different cell lines. Using this approach, we have determined the response of the HGPRTase activity of communication-competent and communication-incompetent cells to changes in PRPP content. The HGPRTase activity of HGPRT+ communication-competent NS cells responds to changes of their own PRPP level, as well as to changes of the PRPP level of HGPRT- cells with which they are co-cultured. In contrast, the HGPRTase activity of the HGPRT+, but communication-incompetent L929 cells responds to changes of their own PRPP content but not to changes of the PRPP content of the cocultured HGPRT- cells. These and other experiments show that PRPP is freely exchangeable between communication-competent cells and that the intracellular activity of HGPRTase in one cell can be regulated by changes in the levels of its substrate in another cell through metabolic cooperation. The results also indicate that HGPRTase normally functions at a small fraction of its total activity, and that this can be greatly increased by raising the intracellular PRPP levels. Furthermore, it is found that when communication-competent cells establish intercellular communication, they share a common pool of PRPP and of purine nucleotides. This approach can be used as the basis of a biochemical method for the quantitation of metabolic cooperation between cells.     

Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis

BACKGROUND: Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are well-recognized antiparasitic drug targets. HGPRT is also a paradigmatic representative of the phosphoribosyltransferase family of enzymes, which includes other important biosynthetic and salvage enzymes and drug targets. To better understand the reaction mechanism of this enzyme, we have crystallized HGPRT from the apicomplexan protozoan Toxoplasma gondii as a ternary complex with a substrate and a substrate analog. RESULTS: The crystal structure of T. gondii HGPRT with the substrate Mg2+-PRPP and a nonreactive substrate analog, 9-deazaguanine, bound in the active site has been determined at 1.05 A resolution and refined to a free R factor of 15.4%. This structure constitutes the first atomic-resolution structure of both a phosphoribosyltransferase and the central metabolic substrate PRPP. This pre-transition state complex provides a clearer understanding of the structural basis for catalysis by HGPRT. CONCLUSIONS: Three types of substrate deformation, chief among them an unexpected C2'-endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-pi interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.     

Guanine salvage by organ cultures of mouse tooth germs

The effect of mycophenolic acid (MPA) which inhibits the biosynthesis of guanosine monophosphate (GMP) in organ cultures of mouse tooth germs can be partially counteracted by adding guanine to the MPA cultures. This may be due to salvaging guanine by the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), or to competition for a common membrane carrier involved in mediated transport of both guanine and hypoxanthine in normal biosynthesis and also of MPA. Experiments were carried out to compare the effect of either hypoxanthine or guanine on the MPA-caused inhibition. While addition of guanine to the MPA cultures (MPAG) supports growth equal to controls and development of dental-enamel junction (DEJ) to a level intermediate between control and MPA the addition of hypoxanthine (MPAHX) supports growth and DEJ development not better than MPA. This indicates that guanine is salvaged by HGPRT to GMP while hypoxanthine, salvaged to inosinic acid (inosinic monophosphate, IMP) is ineffective because the MPA inhibition is on the pathway from IMP to GMP.     

Thermal stability of Artemia HGPRT: effect of substrates on inactivation kinetics

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, E.C. 2.4.2.8) from Artemia cysts exhibits maximum activity at 70°C. Its thermal stability has been examined following enzymatic activity as a function of temperature. Cold-induced renaturation experiments of samples heated at increasing temperatures showed that reversibility of thermal inactivation depends on the incubation time and final temperature. Prolonged incubation of the thermoinactivated enzyme at 0°C did not afford any further increase of the catalytic activity at 37°C. The complex substrate PRPP:Mg protects HGPRT from thermal inactivation. However, incubations with hypoxanthine rendered a less thermostable enzyme at any temperature tested. The irreversible inactivation of HGPRT proceeds in two exponential steps. The analysis of the apparent rate constants for the fast and the slow phases, λ₁ and λ₂ as per the Lumry and Eyring model suggests the existence of more than three states in the thermal denaturation pathway of the free enzyme. In the presence of PRPP:Mg the irreversible process follows a single exponential and proceeds very slowly below 70°C. PRPP:Mg also protects the enzyme from inactivation by NEM and pCMB, suggesting that -SH groups may be in the vicinity of the active site     

The separation of adenine and hypoxanthine-guanine phosphoribosyl transferases isoenzymes by disc gel electrophoresis

Hypoxanthine-guanine (HGPRT; E.C. 2.4.2.8) and adenine (APRT; E.C. 2.4.2.7) phosphoribosyl transferases were studied by disc electrophoresis on polyacrylamide gel. The positions of the isoenzymes were detected by radiochemical enzyme assay. The nucleotide products of the reactions were precipitated in the gel with lanthanum chloride. APRT was found to migrate slightly less rapidly than albumin and produced a single narrow symmetrical peak of activity. HGPRT migrated 25–50% more slowly than albumin and produced a broad zone of activity consisting of four unequal peaks. The APRT enzyme of Rhesus monkey liver and the HGPRT enzyme of sheep erythrocytes migrated notably slower than the corresponding human enzymes. An isoenzyme of APRT was detected in human erythrocytes which migrated more rapidly than that of most individuals. In all instances, the adenine was utilized by one electrophoretic component and hypoxanthine and guanine by another. Furthermore, the components which utilized hypoxanthine and guanine were inseparable. The sensitivity of the assay made it possible to assess the electrophoretic and enzymatic characteristics of HGPRT isoenzymes on aliquots of hemolysates capable of producing 0.5 picomoles of IMP per minute. In human erythrocytes with normal enzyme content, this amount of activity is present in approximately 50 nanoliters of cells.Aided by U.S. Public Health Service grants Nos. HD 04608 and HD 03015 from the National Institute of Child Health and Human Development, National Institutes of Health.     

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.     

Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor

The gene for Escherichia coli guanine-xanthine phosphoribosyltransferase was placed after the high efficiency lambda phage leftward promoter in plasmid pHEGPT also containing the lambda CI857 temperature-sensitive repressor. Guanine-xanthine phosphoribosyltransferase increases 780-fold when cells containing pHEGPT are shifted from 30 to 42 degrees C. Guanine-xanthine phosphoribosyltransferase represents approximately 5% of the protein in a crude extract of induced cells. Guanine-xanthine phosphoribosyltransferase may be purified to apparent homogeneity by ammonium sulfate fractionation, Sephadex G-100, and DEAE-cellulose column chromatography. The enzyme has a subunit molecular weight of 18,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a trimer during Sephadex G-100 column chromatography. Guanine-xanthine phosphoribosyltransferase is active from pH 7.5 to 10.5 with maximum activity at pH 9.5. The enzyme is protected from heat inactivation by phosphoribosylpyrophosphate (PRPP). At 65 degrees C, the enzyme has a half-life of 2 min in the absence of PRPP and 90 min in the presence of PRPP. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for guanine, xanthine, hypoxanthine, and PRPP of 2.6, 39, 167, and 95 microM, respectively. The activity of the enzyme with guanine is 2-fold greater than that with xanthine and 3-fold greater than that with hypoxanthine.     

Different substrate specificities of two triazine hydrolases (TrzNs) from Nocardioides species

Nocardioides sp. strain MTD22 degraded atrazine, ametryn and atraton, as did Arthrobacter aurescens strain TC1 and Nocardioides sp. strain C190. These strains contain trzN, a gene coding for TrzN, triazine hydrolase showing a broad substrate range. However, Nocardioides sp. strain AN3 degraded only atrazine despite containing trzN. These differences in s-triazine degradation are presumed to be due to differences in the amino acid sequences of TrzNs. Consequently, 1371 nucleotides of the trzN coding sequences of strains AN3 and MTD22 were determined. Comparisons of the amino acid sequences of TrzNs indicated that three residues of strain AN3 (Thr(214), His(215) and Gln(241)) were distinct from those of the other three strains (Pro(214), Tyr(215) and Glu(241)). To confirm the relationships between these amino acid sequences and the substrate specificities of TrzNs, wild and chimera trzN genes were constructed and expressed in Escherichia coli cells. Cells expressing wild MTD22 trzN (Pro(214)Tyr(215)Glu(241)) and chimera AN3-MTD22 trzN (Thr(214)His(215)Glu(241)) degraded all s-triazines, but the degradation rate was markedly decreased in AN3-MTD22 trzN. Wild AN3 trzN (Thr(214)His(215)Gln(241)) and chimera MTD22-AN3 trzN (Pro(214)Tyr(215)Gln(241)) degraded only atrazine. These results suggest that the substitution of Glu(241) for Gln(241) significantly decreases enzyme affinity for ametryn and atraton.     

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin fibroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex.

The crystal structures of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been determined at 1.65 and 1.90 A resolution. These complexes, which crystallize in space groups P2(1) (a = 65.45 A, b = 90.84 A, c = 80. 26 A, and beta = 92.53 degrees ) and P2(1)2(1)2(1) (a = 84.54 A, b = 102.44 A, and c = 108.83 A), each comprise a tetramer in the crystallographic asymmetric unit. All active sites in the tetramers are fully occupied by the nucleotide. Comparison of these structures with that of the xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex reported in the following article [Héroux, A., et al. (1999) Biochemistry 38, 14495-14506] shows how T. gondii HGPRT is able to recognize guanine, hypoxanthine, and xanthine as substrates, and suggests why the human enzyme cannot use xanthine efficiently. Comparison with the apoenzyme reveals the structural changes that occur upon binding of purines and ribose 5'-phosphate to HGPRT. Two structural features important to the HGPRT mechanism, a previously unrecognized active site loop (loop III', residues 180-184) and an active site peptide bond (Leu78-Lys79) that adopts both the cis and the trans configurations, are presented.     

Clinical and Biochemical Manifestations and Molecular Characterization of the Mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5‐phosphoribosyl‐1‐pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch–Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).