prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase

• Published in: Journal of Bacteriology Vol. 173; no. 6; pp. 1978 - 1986
• Main Authors: Post, D.A, Switzer, R.L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.03.1991
• Subjects: Alleles; Bacteriology; Biological and medical sciences; Cations, Divalent; Chromosome Mapping; Cloning, Molecular; enzyme activity; Fundamental and applied biological sciences. Psychology; Hot Temperature; kinases; Kinetics; Metabolism. Enzymes; Microbiology; Molecular Weight; mutants; Mutation; Phenotype; Restriction Mapping; ribose-phosphate pyrophosphokinase; Ribose-Phosphate Pyrophosphokinase - antagonists & inhibitors; Ribose-Phosphate Pyrophosphokinase - genetics; Salmonella typhimurium; Salmonella typhimurium - genetics; structural genes; Purification; Enzyme; Salmonella typhimurium; Characterization; Allele; Enzymatic activity; Gene; Bacteria; Mutation; Kinetic parameter; Molecular cloning; Ribosephosphate pyrophosphokinase; Enterobacteriaceae
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/jb.173.6.1978-1986.1991

The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N. K. Pandey and R. L. Switzer, J. Gen. Microbiol. 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis.