Anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl-transferase from Salmonella typhimurium. Inactivation of glutamine-dependent anthranilate synthetase by agarose-bound anthranilate.
Exposure of the anthranilate synthetase-anthranilate phosphoribosyltransferase enzyme complex (chorismate pyruvate-lyase (amino-accepting) EC 4.1.3.27 and N-(5-phosphoribosyl)-anthranilate pyrophosphate phosphoribosyl-transferase, EC 2.4.2.18) from Salmonella typhimurium to agarose-bound anthranilate led to the slow inactivation of glutamine-dependent anthranilate synthetase activity, an activity dependent on protein-protein interaction in the enzyme complex. Region I of phosphoribosyltransferase, the location of the enzyme complex glutaminase activity, is the site of alteration. Phosphoribosyltransferase and NH3-dependent anthranilate synthetase activities and trypto phan regulation of phosphoribosyltransferase were unaffected by the anthranilate matrix. The molecular weight (280 000) and subunit molecular weight (62 000) of the enzyme complex eluted from an anthranilate matrix were identical to those of enzyme complex purified by classical methodology. The enzyme complex could be partially protected against inactivation by storiing in 0.1 M L-glutamine or 30% glycerol and completely protected by storing in 50% glycerol at -18 degrees C. Evidence is presented that the anthranilate matrix acts as a hydrophobic matrix and may be binding to and altering a hydrophobic region in the enzyme complex. The anthranilate matrix provides a convenient tool for altering a specific region of an enzyme complex without covalent modification. At the same time, the results demonstrate that affinity matrices are not necessarily innocuous but may subject macromolecules to an adverse environment not previously recognized.
D000495:Allosteric Site; D000877:Anthranilate Phosphoribosyltransferase / Q000378:metabolism*; D000878:Anthranilate Synthase / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005973:Glutamine / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010430:Pentosyltransferases / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D012685:Sepharose; D012995:Solubility; D062367:ortho-Aminobenzoates / Q000494:pharmacology*
D009097:Multienzyme Complexes; D062367:ortho-Aminobenzoates; D005973:Glutamine; D012685:Sepharose; D010430:Pentosyltransferases; D000877:Anthranilate Phosphoribosyltransferase; D000878:Anthranilate Synthase