Studies on the regulation in vivo and structure/function relationships of the enzyme 5-phosphoribosyl-alpha-1-pyrophosphate synthetase from Salmonella typhimurium
Post, David A.
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https://hdl.handle.net/2142/20984
Description
Title
Studies on the regulation in vivo and structure/function relationships of the enzyme 5-phosphoribosyl-alpha-1-pyrophosphate synthetase from Salmonella typhimurium
Author(s)
Post, David A.
Issue Date
1992
Doctoral Committee Chair(s)
Switzer, Robert L.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Little is known about the amino acids present in the active site of the enzyme phosphoribosylpyrophosphate (PRPP) synthetase or the regulation of the structural gene (prs) in vivo. Site directed mutagenesis and characterization of mutant proteins were used to examine possible residues involved in catalysis. Initial studies of the transcriptional regulation of the prs gene were also conducted.
The Salmonella typhimurium prsB mutant was reported to produce undetectable levels of PRPP synthetase activity (Pandey, N. K., and R. L. Switzer, J. Gen. Microbiol. 128: 1863-1871, 1982). It was shown that prsB was an allele of the prs gene, therefore it was renamed prs-100. The prs-100 mutation was the replacement of Arg-78 by Cys. The mutant PRPP synthetase was purified and determined to possess elevated Michaelis constants, a reduced maximal velocity (30% of wild type) and reduced sensitivity to the allosteric inhibition. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations.
Histidine 130 (His130) was proposed to have a role in divalent cation binding (Bower et al., J. Biol. Chem. 264: 10287-10291, 1989). To investigate this, His130 was replaced by glutamine, tyrosine, asparagine and aspartate by site directed mutagenesis. The mutant proteins were over-produced, purified and characterized. The mutant enzymes had altered Michaelis constants and maximal velocities. It was demonstrated that His130 was not involved in divalent cation binding, but was crucial for catalysis.
The prs leader is 417 bp in S. typhimurium. The region between the prs coding region and an upstream gene (hemA) in S. typhimurium was cloned and sequenced. This region was shown to encode two ORFs of unknown function, which were transcribed in the same direction as the prs gene. Northern blots showed that the prs message is transcribed from two promoters, the first promoter (P$\sb1$) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P$\sb2$), located within the ORF 2 coding frame, which transcribes the prs gene only.
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