Anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferases from Salmonella typhimurium. Purification of the enzyme complex and analysis of multiple forms.
1. The anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme complex (chorismate pyruvatelyase (amino-accepting), EC 4.1.3.27) - (N-(5'-phosphoribosyl)-anthranilate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.18), from Salmonella typhimurium has been purified with high yields to homogeneity. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme complex revealed one major band containing 96% of the protein. The final yield of enzyme complex activity ranged from 30 to 60%. The absorbance spectrum of enzyme complex showed a peak at 280 nm and fine structure with peaks at 253, 259, 266 and 269 nm. These latter wavelengths correspond closely with the known absorbance maxima of phenylalanine. 2. When purified enzyme complex was subjected to standard gel electrophoresis, a four band pattern of protein peaks was consistently observed. The major enzyme complex band was apparently the native tetramer, having a molecular weight 280 000 and containing ammonia- and glutamine-dependent anthranilate synthase activity. The other three bands were molecular weight isomers of the major enzyme complex band. Two forms of molecular weight isomers were present: dimers and an aggregate of the native enzyme complex. The molecular weight isomers of the enzyme complex may represent forms generated by aggregation and denaturation of the native enzyme complex. 3. A new and highly sensitive spectrophotometric assay for phosphoribosyl-transferase is described. The method is based upon the difference in extinction coefficients between anthranilate and N-(5'-phosphoribosyl)anthranilate.
D000877:Anthranilate Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D000878:Anthranilate Synthase / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D010430:Pentosyltransferases / Q000302:isolation & purification*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010430:Pentosyltransferases; D000877:Anthranilate Phosphoribosyltransferase; D000878:Anthranilate Synthase