Metal ion requirement and tryptophan inhibition of normal and variant anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complexes from Salmonella typhimurium

Abstract

1. 1.|Both Mn 2+ and Co 2+ can replace Mg 2+ 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 phosphoribosyltransferase, EC 2.4.2.18) from Salmonella typhimurium. They have much lower, apparent K m values than Mg 2+, both for glutamine-dependent anthranilate synthase ( Mn 2+ = 1.1 μM , Co 2+ = 2.6 μM , Mg 2+ = 83 μM ) and for phosphoribosyltransferase ( Mn 2+ = 16 μM , Co 2+ = 14.6 μM, Mg 2+ = 133 μM ). The ratio of total Mg 2+ to total Mn 2+ found in a cell extract of S. typhimurium trpE2, the source of normal enzyme complex, was found to be 350, suggesting that Mg 2+ is probably utilized by the enzyme complex in vivo under our growth conditions. 2. 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 ( K i = 0.4 μM compared to K i = 20 μM 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. 3.|In the variant enzyme complex the apparent K m for Mg 2+ is elevated to 360 μM for glutamine-linked anthranilate synthase but reduced to 75 μM for phosphoribosyltransferase. 4. 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.