Metal ion requirement and tryptophan inhibition of normal and variant anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complexes from Salmonella tyrhimrium.
1. Both Mn2+ and Co2+ can replace Mg2+ as the required divalent cation for all activities of the enzyme complex between anthranilate synthase (chorismate pyruvate-lyase (amino-accepting), EC 4.1.3.27) and anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (N(5'-phosphoribosyl)-anthranilate:pyrophosphate phosphoribosytransferase, EC 2.4.2.18) from Salmonella typhimurium. They have much lower apparent Km values than Mg2+, both for glutamine-dependent anthranilate synthase (Mn2+ = 1.1 muM, Co2+ - 2.6 muM, Mg2+ = 83 muM) and for phosphoribosyltransferase (Mn2+ = 16 muM, Co2+ = 14.6 muM, Mg2+ = 133 muM). The ratio of total Mg2+ to total Mn2+ found in a cell extract of S. typhimurium trpE2 , the source of normal enzyme complex, was found to be 350, suggesting that Mg2+ is probably utilized by the enzyme complex in vivo under our growth conditions. 2. An enzyme complex has been isolated from a mutant strain of S. typhimurium (SO-515) that has a variation in the anthranilate synthase subunit which is thought to be a single amino acid substitution. This variation causes glutamine-dependent anthranilate synthase to be hypersensitive to feedback inhibition by tryptophan (Ki = 0.4 muM compared to Ki = 20 muM for normal enzyme complex). The phosphoribosyltransferase in the variant enzyme complex is also hypersensitive to tryptophan but the kinetics are complex and involve activation by tryptophan in the presence of low amounts of 5-phosphoribosyl 1-pyrophosphate. 3. In the variant enzyme complex the apparent Km for Mg2+ is elevated to 360 muM for glutamine-linked anthranilate synthase but reduced to 75 muM for phosphoribosyltransferase. 4. These results suggest that the variant enzyme complex has altered tertiary and quaternary structures and that regulation of both activities is effected by tryptophan binding to only anthranilate synthase.
D000877:Anthranilate Phosphoribosyltransferase / Q000378:metabolism*; D000878:Anthranilate Synthase / Q000378:metabolism*; D003035:Cobalt / Q000494:pharmacology*; D004789:Enzyme Activation; D014644:Genetic Variation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010430:Pentosyltransferases / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013045:Species Specificity; D014364:Tryptophan / Q000494:pharmacology*
D009097:Multienzyme Complexes; D003035:Cobalt; D008345:Manganese; D014364:Tryptophan; D010430:Pentosyltransferases; D000877:Anthranilate Phosphoribosyltransferase; D000878:Anthranilate Synthase; D008274:Magnesium