Overproduction disease in man due to enzyme feedback resistance mutation. Purine overproduction in gout due to excessive activity of mutant feedback-resistant phosphoribosylpyrophosphate synthetase.

Physiologically superactive phosphoribosylpyrophosphate (PRPP) synthetase, due to feedback resistance mutation, was found in a family with excessive purine production, gout and uric acid lithiasis. The superactivity of the mutant enzyme was manifest in the propositus' erythrocytes and cultured fibroblasts, in increased generation, content and metabolic availability of PRPP, leading in the fibroblasts to acceleration of the rate of purine synthesis de novo. One of the propositus' two siblings was similarly affected, but the propositus' father, his second brother and four sons, were all clinically and biochemically normal. The mother was clinically normal and normouricemic, but hyperuricosuric. Cultured fibroblasts from her skin exhibited variability in PRPP content and availability and in the rate of purine synthesis de novo. The mother's cultures were found to contain a mosaicism of two cell populations, one with normal and the other with mutant PRPP synthetase, indicating an X-linked pattern of inheritance of the PRPP synthetase abnormality in this gouty family.     

The mode of genetic transmission of a gouty family with increased phosphoribosylpyrophosphate synthetase activity

Summary The mode of genetic transmission of gout and increased activity of phosphoribosylpyrophosphate synthetase (PRPPS) was studied in one family. Among 15 members of Family F, two male members had gout and had PRPPS activity of erythrocyte lysates three times higher than normal subjects. Five female members had activity 2.5 times higher than normal. The difference between the activities of male and female affected members was statistically significant (P<0.05). To examine the genetic trait of this abnormal PRPPS, the incorporation of ³H-adenine into erythrocytes or lymphocytes was studied using autoradiography. The number of grains which show the uptake of labeled adenine into cells revealed a normal distribution pattern in two normal persons and in two male patients, and a mixed pattern of the two cell populations in two female affected members. These results suggested mosaicism in female members and X-linked dominant transmission of this trait. Thermal inactivation of PRPPS of an affected female was intermediate between that from a normal subject and that from the affected males. This result showed the heterogeneity of the PRPPS from the hemolysate of an affected famale. The genotype of PRPPS on the X-chromosome was assumed and the lod score between PRPPS and Xg was also estimated. From these findings and electrophoretical study, it was suggested that the abnormal enzyme was a mutant enzyme transmitted in an X-linked dominant trait, and that the mutation occurred on the structural gene of the PRPPS.     

Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purin auxotroph by a procedure designed to select guanosine-utilizing mutants. Strain GP122 had roughly 15% of the PRPP synthetase activity and 25% of the PRPP pool of its parent strain. The mutant exhibited many of the predicted consequences of a decreased PRPP pool and a defective PRPP synthetase enzyme, including: poor growth on purine bases; decreased accumulation of 5-aminoimidazole ribonucleotide (the substrate of the blocked purE reaction) under conditions of purine starvation; excretion of anthranilic acid when grown in medium lacking tryptophan; increased resistance to inhibition by 5-fluorouracil; derepressed levels of aspartate transcarbamylase and orotate phosphoribosyltransferase, enzymes involved in the pyrimidine de novo biosynthetic pathway; growth stimulation by PRPP-sparing compounds (e.g. guanosine, histidine); poor growth in low phosphate medium; and increased heat lability of the defective enzyme. This mutant strain also had increased levels of guanosine 5'-monophosphate reductase. This genetic lesion, designated prs, was mapped by conjugation and phage P22-mediated transduction at 35 units on the Salmonella linkage map.     

Phenylalanyl synthetase function in cultured fibroblasts from subjects with progeria.

Since progeria cells contain a diversity of altered proteins, some aspects of phenylalanyl synthetase function were examined in semipurified extracts of cultured skin fibroblasts using mixed rabbit tRNA as acceptor. No significant differences were found in the Km and Vmax for phenylalanine or ATP in progeria cells compared with controls. Initial velocities of both progeria and control synthetases were lower at late passage owing to either reduced enzyme content or reduced catalytic efficiency. Reverse phase 5 chromatography of tRNAs acylated by progeria and control synthetases gave a single peak of labeled phenylalanine tRNA in all cases with no secondary peaks evident. Total activity of phenylalanyl synthetase in progeria cells was similar to that of control cells at early passage while late-passage control cells had lower specific activities of these synthetases per unit protein.     

Characterization of purine nucleotide metabolism in cultured fibroblasts with deficiency of hypoxanthine-guanine phosphoribosyltransferase and with superactivity of phosphoribosylpyrophosphate synthetase

Cultured fibroblasts with hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency exhibited acceleration of purine synthesis de novo, absence of salvage IMP synthesis from hypoxanthine, but normal total IMP synthesis. Cells with phosphoribosylpyrophosphate synthetase superactivity exhibited acceleration of both de novo and salvage IMP synthesis and increased total IMP synthesis. The study of mutant cells furnished evidence that in normal as well as mutant cells, GMP and AMP are not converted to each other in significant amounts and that these nucleotides are not degraded by nucleotidases. Purine nucleotide degradation in fibroblasts occurs mainly by dephosphorylation of IMP. In HGPRT-containing cells, salvage IMP synthesis from preformed and exogenously supplied hypoxanthine is the main source for IMP production.     

Increased phosphoribosylpyrophosphate synthetase activity in fibroblasts of hypoxanthine-guanine phosphoribosyl transferase deficient patients.

An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This eccessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.     

Studies of the quaternary structure and the chemical properties of phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

Phosphoribosylpyrophosphate (PRPP) synthetase (EC 2.7.6.1) was purified to virtual homogeneity from Salmonella typhimurium cells by a modification of previously published procedures. The molecular weight of the subunit was determined to be 31,000 +/- 3,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and sedimentation equilibrium analysis of the enzyme dissolved in 6 M guanidine hydrochloride. The amino acid composition of the enzyme was determined. Proline was identified as the only NH2-terminal residue. PRPP synthetase is apparently composed of identical or nearly identical subunits. NATIVE PRPP synthetase exists in multiple states of aggregation under all conditions. However, two predominant states were demonstrated under certain conditions. A form with molecular weight of 320,000 +/- 20,000 was found at pH 7.5 in the presence of MgATP. At pH 8.2 to 8.6, with or without MgATP, the predominant form corresponded to a molecular weight of 150,000 to 200,000; sedimentation equilibrium and velocity analysis indicated 160,000 +/- 15,000 as the most reliable molecular weight. More highly aggregated forms were observed at 4 degrees and higher protein concentrations. Removal of inorganic phosphate from PRPP synthetase by dilution or dialysis resulted in disaggregation. The fundamental unit of PRPP synthetase appears to consist of five (or possibly six) subunits, which can associate to form a dimer (10 or 12 subunits) and more highly aggregated forms. A pentameric subunit structure is consistent with the multiple species resolved by electrophoresis of the native enzyme in discontinuous polyacrylamide gel systems. Visualization of PRPP synthetase by negative staining with uranyl acetate and electron microscopy revealed fields of very asymmetric molecules, the dimensions of which corresponded to the M = 160,000 form. Dimers and higher aggregates of this unit were also seen. An unusual model, in which the five subunits are asymmetrically arranged, accounts very well for the electron microscopic appearance of the enzyme. The tendency of the enzyme to aggregate is viewed as a consequence of the unsatisfied bonding regions of the fundamental asymmetric unit.     

Evidence for a defect of holocarboxylase synthetase activity in cultured lymphoblasts from a patient with biotin-responsive multiple carboxylase deficiency.

We report here the expression of biotin-responsive multiple carboxylase deficiency in cultured lymphoblasts of a patient whose fibroblasts belong to the bio genetic complementation group. Cultured lymphoblasts from the patient lost propionyl-CoA carboxylase (PCC) and beta-methylcrotonyl-CoA carboxylase (MCC) activities at a faster rate than normal cells when grown in biotin-deficient medium. Recovery of normal PCC and MCC activities, which was independent of protein synthesis, required a 2,500-fold higher biotin concentration than that required by normal lymphoblasts. Holocarboxylase synthetase activity was detected in cell-free extracts through the biotinylation of endogenous apo-PCC in the presence of ATP to form active holo-PCC. While the apo-PCC in extracts of normal biotin-starved lymphoblasts could be activated to 28% of maximal activity, extracts of patient lymphoblasts did not exhibit any ATP and biotin-dependent increase in PCC activity. A normal cell extract, cleared of apocarboxylases by immunoprecipitation, stimulated the PCC activity of a patient cell extract 20-fold. These results indicate that the apoenzyme in bio cells is normal and that the defect lies in the holocarboxylase synthetase.     

Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis

The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+) and Cu(2+) but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+) and Cu(2+) ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design.     

Oxidative DNA damage in cultured fibroblasts from patients with hereditary glutathione synthetase deficiency

The SH compound glutathione (GSH) is involved in several fundamental functions in the cell, including protection against reactive oxygen species (ROS). Here, we studied the effect on oxidative DNA damage in cultured skin fibroblasts from patients with hereditary GSH synthetase deficiency. Our hypothesis was that GSH-deficient cells are more prone to DNA damage than control cells. Single cell gel electrophoresis (the comet assay) in combination with the formamidopyrimidine DNA glycosylase enzyme, which recognizes oxidative base modifications, was used on cultured fibroblasts from 11 patients with GSH synthetase deficiency and five control subjects. Contrary to this hypothesis, we found no significant difference in background levels of DNA damage between cells from patients and control subjects. To study the induction of oxidative DNA damage without simultaneous DNA repair, the cells were γ-irradiated on ice and DNA single-strand breaks measured. The patient and control cells were equally sensitive to induction of single strand breaks by γ-irradiation. Therefore, factors other than GSH protect DNA from oxidative damage. However, cells with a high background level of oxidative DNA damage were found to be more sensitive to ionizing radiation. This suggests that differences in background levels of oxidative DNA damage may depend on the cells' intrinsic protection against induction of oxidative damage.     

prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase.

The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N.K. Pandey and R.L. Switzer, J. Gen. Microbiol, 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis.     

Structural analysis of normal and mutant insulin receptors in fibroblasts cultured from families with leprechaunism.

Leprechaunism is an inherited disorder characterized by insulin resistance and intrauterine growth restriction. In this study we analyze insulin binding and subunit structure of the insulin receptor in dermal fibroblasts cultured from three unrelated families whose probands (Ark-1, Atl, and Minn) were affected by leprechaunism. Cells cultured from all three probands had markedly reduced insulin binding at equilibrium. Fibroblasts cultured from the parents of Ark-1 and Atl had partial and differing degrees of impairment in insulin binding. The structure of the alpha subunit of insulin receptors was analyzed by cross-linking 125I-insulin to plasma membranes. A major band of 350 kilodaltons (kD) (corresponding to the heterotetrameric insulin receptor alpha 2 beta 2) was observed in control and leprechaun fibroblasts. The relative amount of radioactivity cross-linked to plasma membranes reflected the genetic variations seen in insulin binding to intact cells. In reducing gels, 125I-insulin was cross-linked equally to a 250-kD (alpha-alpha dimer) and a 125-kD (alpha monomer) protein in cells from controls, the parents of Ark-1 and Atl, and probands Atl and Minn. By contrast, cells from the Ark-1 proband had diminished cross-linking of alpha-alpha dimers. The ratio of dimer to monomer in cells from controls was 0.93 +/- 0.06, and that in cells from Ark-1 was 0.31 +/- 0.19 (P less than .01). Beta-subunit structure and function was analyzed by studying insulin-enhanced autophosphorylation. Although maximal stimulation of beta-subunit phosphorylation was reduced to 30% in proband Ark-1 fibroblasts, this reduction was quantitatively related to reduced insulin binding. These results indicate that mutations causing severe insulin resistance and defective insulin binding are transmitted with autosomal recessive patterns of inheritance and that heterogeneity exists for these mutations. The mutation in pedigree Ark-1 most likely produces conformational changes in alpha-subunit interaction.     

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

Molecular Genetics and Genomics - In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes...