THE GENETIC AND FUNCTIONAL BASIS OF PURINE NUCLEOTIDE FEEDBACK-RESISTANT PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE SUPERACTIVITY

The genetic and functional basis of phosphoribosylpyrophosphate synthe tase (PRS) superactivity associated with purine nucleotide inhibitor-r esistance was studied in six families with this X chromosome-linked pu rine 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, demonst rated that each PRS1 cDNA contained a distinctive single base substitu tion predicting a corresponding amino acid substitution in the PRS1 is oform, Overall, the array of substitutions encompassed a substantial p ortion of the translated sequence of PRS1 cDNA. Plasmid-mediated expre ssion of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yield ed recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance co mparable to that found in cells of the respective patient, Kinetic ana lysis of recombinant mutant PRS1s showed that widely dispersed point m utations in the X chromosome-linked PRPS1 gene encoding the PRS1 isofo rm result in alteration of the allosteric mechanisms regulating both e nzyme inhibition by purine nucleotides and activation by inorganic pho sphate, The functional consequences of these mutations provide a tenab le basis for the enhanced production of phosphoribosylpyrophosphate, p urine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.