Selective expression of phosphoribosylpyrophosphate synthetase superactivity in human lymphoblast lines.

Journal of Biological Chemistry

Taylor

Smith

et al. 1996

To define the enzymatic and genetic basis of X-linked phosphoribosylpyrophosphate synthetase (PRS) catalytic superactivity, we measured concentrations of Xlinked PRS1 and PRS2 isoforms in cultured fibroblasts and lymphoblasts by immunoblotting after separation by polyacrylamide-urea isoelectric focusing. PRS1 comprised >80% of measurable PRS isoforms in all fibroblast strains, but PRS1 concentrations in cells from six affected males exceeded those in normal cells by 2-6-fold. PRS absolute specific activities (activity per mg of PRS isoforms) were comparable in all fibroblast strains and in purified recombinant normal PRS1, confirming selectively increased levels of PRS1 isoform as the enzymatic basis of PRS catalytic superactivity. Cloning, sequencing, and expression of normal subject-and patient-derived PRS cDNAs predicted normal translated region sequences for both PRS isoforms and revealed no differences in catalytic properties of recombinant PRS1. Normal and patient PRPS1 transcribed but untranslated DNA sequences were also identical. Northern blot analysis showed selective increase in relative concentrations of PRS1 transcripts in patient fibroblasts. In PRS catalytic superactivity, overexpression of the normal PRS1 isoform thus appears to result from an altered pretranslational mechanism of PRPS1 expression. In lymphoblasts, however, expression of this alteration is attenuated, explaining the absence of phenotypic expression of PRS catalytic superactivity in these cells. Superactivity of phosphoribosylpyrophosphate (PRPP)1 synthetase (EC 2.7.6.1; PRS) is an X-linked human disorder (1) characterized by purine nucleotide and uric acid overproduction (2-4), gout (2, 5), and, in some families, neurodevelopmental impairment (5-8). Accelerated production of the purineregulatory substrate PRPP provides a unitary pathogenetic mechanism for excessive synthesis of purine nucleotides and, ultimately, uric acid in affected individuals (3, 4, 9). Nevertheless, diversity is apparent in the aberrant enzymatic properties resulting in PRS superactivity in different families (3,5,8). Defective allosteric regulation of PRS activity by purine nucleotide inhibitors (such as ADP and GDP) and by the activator P i characterizes one kinetically defined class of superactive PRS (8). Defects in allosteric regulation of PRS result from point mutations in the translated sequence of PRPS1 (8, 10), one of the two X-linked genes (PRPS1 and PRPS2) (11, 12) encoding independently active (13) and highly homologous (14, 15) PRS isoforms (PRS1 and PRS2, respectively).In a second and, in our experience, more frequently encountered class of PRS superactivity, regulation of enzyme activity by nucleotide inhibitors is normal, as are affinities for substrates and activators (Mg 2ϩ , P i ) (16 -19). Increased maximal reaction velocity (V max ) is the only identifiable kinetic alteration in the PRS of such individuals (catalytic superactivity) (20). Although indirect evidence for structural alterations in PRS has been reported in catalytic supe...

Section: Discussionsupporting

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Superactivity Of Phosphoribosylpyrophosphate (Prpp)mentioning

confidence: 99%

mentioning

confidence: 99%

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Overexpression of the Normal Phosphoribosylpyrophosphate Synthetase 1 Isoform Underlies Catalytic Superactivity of Human Phosphoribosylpyrophosphate Synthetase

Journal of Biological Chemistry

Taylor

Smith

et al. 1996

“…Kinetic defects underlying PRS superactivity include: (a) regulatory defects characterized by purine nucleotide inhibitor-resistance, sometimes associated with increased apparent affinity of PRS for Pi (9,(11)(12)(13)(14); (b) catalytic defects in which maximal reaction velocity is increased but substrate and activator affinities and inhibitor responsiveness are normal (15)(16)(17)(18)(19); (c) combined regulatory and catalytic defects (20,21); and (d) increased affinity for the substrate Rib-5-P (22). Although regulatory defects comprise all or a part of the kinetic aberrations resulting in PRS superactivity in only 6 of the nearly 30 affected families studied in detail, infantile or childhood onset and neurodevelopmental impairment accompany hyperuricemia and gout in five of these families (12,13,20,23), but are rare in families with other defects (17,24).…”

Section: Introductionmentioning

confidence: 99%

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

Smith²,

Taylor³

et al. 1995

J. Clin. Invest.

Self Cite

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosomelinked PRPSI gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity. (J. Clin.

Phosphoribosylpyrophosphate synthetase superactivity: A study of five patients with catalytic defects in the enzyme

Losman

Rosenberg

et al. 1986

Arthritis & Rheumatism

Superactive phosphoribosylpyrophosphate (PRPP) synthetases were characterized in fibroblasts and erythrocytes from 5 unrelated men with gout and/or hyperuricemia and uric acid overproduction. The kinetic basis of enzyme superactivity in all patients was increased maximal reaction velocity. Affinities of the enzyimes for substrates and activators and responsiveness to inhibitors were normal, and levels of immunoreactive enzyme in patient and control fibroblast and erythrocyte extracts were comparable. Enzymes purified to homogeneity from 2 patients confirmed the presence of isolated catalytic defects. Altered physical properties of certain of the superactive enzymes suggesteld the presence of several distinctive structural defects among the aberrant forms. Fibroblasts from each affected patient showed increased PRPP concentration :and generation, as well as accelerated rates of all PRPP-requiring purine nucleotide synthetic pathways. Thew findings support the concept that enzyme superactivity results in uric acid overproduction as a consequence of increased rates of PRPP and purine nucleotide synthesis. Cultured cells from female relatives of 2 patients showed evidence for the heterozygous carrier state, as measured both by enzyme activities and by rates of PRPP and purine synthesis. The clinical phenotype in 4 patients was limited to early adult-onset gout and its consequences, whereas the fifth patient expressed a familial constellation of hyperuricemia, sensorineural deafness, ataxia, and renal insufficiency. The severity of the derangements in PRPP synthetase and in PRPP and purine synthesis in cells from the 5 patients, however, was comparable. The neurologic accompaniments of enzyme superactivity found in 1 family described here, and in 2 others described previously, thus may not necessarily be consequences of primary defects in PRPP synthetase.Superactivity of phosphoribosylpyrophosphate (PRPP) synthetase, the enzyme which catalyzes the synthesis of the purine regulatory substrate, PRPP, from adenosine triphosphate (ATP) and ribose-5-P (I), has been associated with gout in nearly one dozen families (2-1 1). A causal relationship between excesses of enzyme activity, inherited as an X chromosome-linked trait (12-14), and increased rates of purine nucleotide and uric acid production has been established from detailed studies of affected members of some of these families (4)(5)(6)8,(14)(15)(16)(17). Erythrocytes and fibroblasts from these individuals show increased concentrations and rates of generation of 8,[15][16][17], and increased rates of purine nucleotide synthesis de novo are demonstrable both in vivo (8,16,(18)(19)(20) and in cultured fibroblasts from these patients (3)(4)(5)8,16,17). These findings indicate that excessive purine nucleotide production and uric acid excre-