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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 chromosome-linked PRPS1 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.