Identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. Evidence for an amino-terminal regulatory site.
The anthranilate synthase-phosphoribosyl transferase complex, a heterotetrameric enzyme made up of the TrpE and TrpD polypeptides, catalyzes three reactions comprising the first two steps of tryptophan biosynthesis in Salmonella typhimurium. All three activities of the complex are subject to feedback inhibition by tryptophan, which results from allosteric effects associated with the binding of one molecule of inhibitor to each of the TrpE subunits of the complex. Random in vitro chemical mutagenesis of the trpE gene was used to generate a collection of mutant forms of the complex which displayed varying degrees of resistance to feedback inhibition. Single amino acid substitutions, identified by DNA sequencing, were found at 14 different residues within the TrpE polypeptide. The residues were distributed throughout TrpE, but those that appeared to be most critical for regulation were found in two clusters, one at the extreme amino-terminal end, including residues Glu-39, Ser-40, and Ala-41, and the other in the middle of the polypeptide, including residues Asn-288, Pro-289, Met-293, Phe-294, and Gly-305. Kinetic and binding studies of the purified mutant complexes demonstrated that 9 of the 14 had a marked decrease in affinity for tryptophan with little or no change in substrate affinity or catalytic capacity. The remaining five enzymes exhibited more subtle changes, having small decreases in inhibitor affinity coupled with small increases in substrate affinity. Mutant enzymes that were not totally feed-back-resistant had a decreased kinetic response to tryptophan binding. All enzymes exhibited alterations in tryptophan-induced conformational changes as monitored by dye-ligand chromatography.
Caligiuri
M G
MG
Department of Biology, University of Virginia, Charlottesville 22901.
Bauerle
R
R
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000877:Anthranilate Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D000878:Anthranilate Synthase / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D003062:Codon; D005246:Feedback; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D016296:Mutagenesis; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism
D000596:Amino Acids; D003062:Codon; D009097:Multienzyme Complexes; D014364:Tryptophan; D000877:Anthranilate Phosphoribosyltransferase; D000878:Anthranilate Synthase