MOLECULAR MECHANISMS OF PRPP-SYNTHETASE OVERACTIVITY

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Phosphoribosylpyrophosphate synthetase (PRPP-synthetase) catalyzes the reaction: Ribose-5-phosphate + Mg2+ PRPP + AMP. The formation of PRPP by this enzyme represents the first step in the de novo synthesis of purines, pyrimidines and pyridines. A considerable amount of information regarding this enzymes' physical and kinetic properties has been amassed. However, very little is known regarding the structural gene encoding PRPP- synthetase or the regulation of its expression. Since PRPP is a key intermediate in the de novo synthesis and salvage pathways of purine metabolism, the enzyme responsible for its synthesis is an important regulator of human purine homeostasis. Supernormal activity of PRPP-synthtase in erythrocytes is assoicated with an X-linked syndrome consisiting of hyperuricemia, hyperuricaciduria, precocious gout and nephrolithiasis. Several of these mutant forms of PRPP-S have been partially characterized. Alterations in kinetic or regulatory properties (or a combination of both) appear to be responsible for the supernormal activity observed in such mutants. The molecular bases for these alterations in catalytic activity remain unknown. Thus, genetic studies of PRPP-synthetase are relevant to improved understanding of normal human purine metabolism as well as the pathogenesis of human disease. The specific aims of this proposal are to: i) isolate and characterize the cDNA encoding normal human PRPP-synthetase; ii) raise monospecific antiserum to human PRPP-synthetase; iii) characterize protein and nucleic acid abnormalities in cells derived from subjects with PRPP-synthetase overactivity; iv) to clone mutant forms of PRPP-synthetase cDNA from subjects with supernormal variants of PRPP-synthetase. The P.I. proposes to isolate PRPP-synthetase cDNA from cDNA libraries by in situ hybridization using oligonucleotide probes. Oligonucleotides will be derived from partial amino acid sequence of peptides obtained from pure enzyme. Overlapping cDNA clones will be sequenced to determine the structure of full-length PRPP-synthetase cDNA from which the complete amino acid sequence will be deduced. The identity of the cDNA will be confirmed by comparison of predicted amino acid sequence to that of peptides which were not the basis for oligonucleotide design and by hybrid selection. cDNA probes obtained in this fashion will then be used to characterize normal and mutant gene expression and to clone mutant PRPP-synthetase cDNAs from lymphocytes and fibroblasts derived from gouty subjects with overactivity of the enzyme. From these studies, enhanced understanding of the molecular basis for this inherited form of gout should result. Furthermore, characterization of a potentially important class of mutations, i.e. those expressed through "supernomal" enzymatic activity should provide insights into the relationship between altered protein structure and abnormal catalytic function.