REGULATION AND MECHANISM OF PRPP SYNTHETASE

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It is proposed to study the metabolic regulation and mechanism of catalysis of the essential biosynthetic enzyme phosphoribosylpyrophosphate (PRPP) synthetase from the enteric bacterium Salmonella typhimurium. This enzyme is of particular interest because (a) PRPP is the first step in a universal, highly branched pathway leading to all nucleotides, two amino acids, and two coenzymes; (b) the enzyme catalyzes an unusual pyrophosphoryl group transfer reaction, and (c) PRPP metabolism is known to play an important role in a number of inherited metabolic diseases leading to anemia or hyperuricemia and gout. It is proposed to clone the structural gene for PRPP synthetase from S. typhimurium and to determine its nucleotide sequence. This will provide the primary structure of the enzyme; active site amino acid residues will be located in the sequence by chemical modification with active site-directed reagents and peptide mapping. These results, combined with previous and proposed mechanistic studies, should provide a much refined picture of enzymic catalysis by PRPP synthetase. The regulation of transcription from the PRPP synthetase gene will be studied by sequencing adjacent control regions and by studying transcription in vitro using DNA isolated on recombinant plasmids. The function of a second gene affecting PRPP synthetase activity will be probed by similar techniques.