MECHANISM OF THE SYNERGISTIC END-PRODUCT REGULATION OF BACILLUS-SUBTILIS GLUTAMINE PHOSPHORIBOSYLPYROPHOSPHATE AMIDOTRANSFERASE BY NUCLEOTIDES

De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An importa nt feature of this inhibition is the fact that certain synergistic nuc leotide pairs give more than additive inhibition, The physiological im portance of synergism is in amplifying regulation by the adenine and g uanine nucleotide end products of de novo synthesis. Using a new metho d to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, a. L. (1978) J, Biol, Chem. 254, 5397-5402] to give strong s ynergistic inhibition of Bacillus subtilis glutamine PRPP amidotransfe rase, An X-ray structure of the ternary enzyme.ADP.GMP complex establi shed that ADP binds to the allosteric A site and GMP to the catalytic C site. GMP increased the binding affinity of ADP for the A site by si milar to 20-fold. Synergism results from a specific nucleotide-nucleot ide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, syn ergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site. Purine base specificity results from a backbone carbonyl interaction of Lys(305') with the 6-NH2 group of ad enine in the A site and a Ser(347) O gamma interaction with the 2-NH2 group of guanine in the C sire. Steric considerations favor binding of the nucleoside diphosphate to the A site. Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism, Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism.