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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK013488-21
Application #
3225068
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-05-01
Project End
1991-04-30
Budget Start
1989-05-01
Budget End
1991-04-30
Support Year
21
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
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Becker, M A; Nosal, J M; Switzer, R L et al. (1994) Point mutations in PRPS1, the gene encoding the PRPP synthetase (PRS) 1 isoform, underlie X-linked PRS superactivity associated with purine nucleotide inhibitor-resistance. Adv Exp Med Biol 370:707-10
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Harlow, K W; Switzer, R L (1990) Sulfhydryl chemistry of Salmonella typhimurium phosphoribosylpyrophosphate synthetase: identification of two classes of cysteinyl residues. Arch Biochem Biophys 276:466-72
Arnvig, K; Hove-Jensen, B; Switzer, R L (1990) Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis. Eur J Biochem 192:195-200
Harlow, K W; Switzer, R L (1990) Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine. J Biol Chem 265:5487-93
Bower, S G; Harlow, K W; Switzer, R L et al. (1989) Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site. J Biol Chem 264:10287-91

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