Feedback regulation in the anthranilate aggregate from wild type and mutant strains of Escherichia coli.

The anthranilate aggregate, which catalyzes the first two reactions of tryptophan biosynthesis in Escherichia coli, consists of two anthranilate synthetase subunits and two phosphoribosyltransferase subunits. The aggregate remains associated under physiological conditions. Three lines of evidence indicate that regulation in the aggregate involves conformational changes associated with the binding of the substrates, chorismate and glutamine, and the feedback inhibitor, tryptophan. First, either chorismate or glutamine, the anthranilate synthetase substrates, can pseudocompetitively reactivate tryptophan-inhibited phosphoribosyltransferase. Second, the regulatory ligands alter the susceptibility of the aggregate to inactivation by metal ions and by the glutamine analog, 6-diazo&oxo-L-norleucine. Third, direct physical evidence for conformational changes was obtained using the fluorescent probe, S-anilino-lnaphthalene sulfonate. The activities of the aggregate are regulated through alterations in the equilibrium between activated and inhibited conformational states associated with ligand binding. Regulation is competitive because the binding of chorismate and tryptopban or glutamine and tryptophan is mutually exclusive for allosteric reasons. Several independent kinetic and inactivation experiments indicate that glutamine binds to the enzyme in the absence of chorismate. However, glutamine hydrolysis does require that chorismate be present. The binding of chorismate is presumably associated with a conformational change required for the hydrolysis of glutamine. Anthranilate synthetase thus has an ordered reaction mechanism, but random substrate binding. Several mutationally altered anthranilate aggregates, either resistant to feedback inhibition, or supersensitive to feedback inhibition, were examined. A double mutant containing both resistant and the supersensitive alleles was very similar in kinetic properties to wild type. Revertants of the supersensitive strain were isolated, and their anthranilate aggregates were analyzed. In one revertant, the co-