Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase activity by end products.

General kinetic properties and inhibition by purine end products of highly purified glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been studied. The enzyme was subject to specific inhibition by the purine nucleotides AMP, ADP, GMP, and GDP. AMP was by far the most effective inhibitor. The action of the inhibitors displayed positive cooperativity and was antagonized by increasing phosphoribosylpyrophosphate concentration. Phosphoribosylpyrophosphate saturation was hyperbolic in the absence of inhibitors (K,,, = 72 + 8 PM), but became positively cooperative in the presence of inhibitors. The concentration of glutamine (Km = 4.3 f 0.4 mM) did not have a large effect on the sensitivity of the enzyme to end product inhibition. Divalent cations were required for both activity and sensitivity to allosteric inhibition. The concentration dependence of these effects and the differential effects of various cations demonstrated that two different cation sites are involved. Three pairs of nucleotide inhibitors, which were weak inhibitors when tested singly, exhibited very pronounced synergistic inhibition; these were: ADP and GMP, ADP and GDP, and GMP and GDP. AMP did not show synergistic inhibition when tested in combination with other nucleotides. The results require the existence of at least one, and possibly two, allosteric nucleotide-binding sites per amidotransferase subunit. The findings in this paper provide a more complete picture of the regulation of de novo purine nucleotide biosynthesis in B. subtilis and a necessary background for the study of the regulation of the oxidative inactivation which this enzyme undergoes in vitro and in vivo.