Metabolic interlock: Mediation of interpathway regulation by divalent cations☆

Abstract In Bacillus subtilis , the focus of the interpathway regulatory relationship between histidine and the tryptophan biosynthetic pathway is anthranilate synthase. This enzyme is composed of two nonidentical subunits, designated E and X , and has an absolute requirement for a divalent cation for activity with either glutamine (amidotransferase), or ammonia (aminase). Tryptophan is an effective in vitro inhibitor of the Mg 2+ -supported enzyme, but in vivo the full potential for inhibition does not appear to be realized. There is, however, a fourfold increase in the apparent K i for tryptophan when Mn 2+ is used, and in the presence of both Mg 2+ and Mn 2+ (100:1, respectively) the inhibition by tryptophan is identical to that observed with Mn 2+ alone. Thus, a partial explanation for the low efficiency of feedback inhibition may be that Mn 2+ is functioning in the in vivo anthranilate synthase reaction. Histidine has no effect on the velocity or the K m for the substrates when Mg 2+ is used to support amidotransferase activity. On the other hand, histidine stimulates the Mn 2+ supported amidotransferase activity two- to fourfold at limiting concentrations of glutamine and chorismic acid. Furthermore, histidine stimulates tryptophan biosynthesis in some genetically derepressed mutants under conditions that implicate a Mn 2+ anthranilate synthase interaction. Thus, histidine increases the rate of tryptophan excretion about fourfold when the intracellular pools of chorismic acid and glutamine are low but has no effect when the pool levels are high. In the presence of both Mg 2+ and Mn 2+ (100:1, respectively) the in vitro activity of the EX complex behaves as if Mn 2+ alone were present, and histidine stimulates activity about threefold. This excellent correlation between the in vivo conditions required to an observed effect of histidine on tryptophan excretion and the in vitro effect of this ligand on the Mn 2+ -supported enzyme support the hypothesis that, in B. subtilis , Mn 2+ is the dominant cation in the anthranilate synthase activity.