Mechanism of the Synergistic End-Product Regulation of Bacillus subtilis Glutamine Phosphoribosylpyrophosphate Amidotransferase by Nucleotides^,

Abstract

De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition. The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis. Using a new method to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, R. L. (1978) J. Biol. Chem. 254, 5397-5402] to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase. An X-ray structure of the ternary enzyme.ADP.GMP complex established 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 approximately 20-fold. Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, synergism 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 Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site. 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.

Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli

“…The cis conformation is highly unusual in any peptide except those preceding a proline residue. An analogous cis peptide has been reported in other PRTases (Henriksen et al, 1996;Somoza et al, 1996;Vos et al, 1997) and in B. subtilis GPATase (Chen et al, 1997). The independent observation of this highly unusual structural feature in the same topological location of several PRTases is consistent with an important role for the cis peptide.…”

“…The dipole of helix 373-387 is oriented to provide additional electrostatic binding energy. A continuum of orientations for nucleotide binding to the PRPP loop of PRTases has been reported (Eads et al, 1994;Scapin et al, 1994;Smith et al, 1994;Schumacher et al, 1996;Somoza et al, 1996;Chen et al, 1997). The orientations differ by rotation of the nucleotide about its 5'-phosphate in the fiist turn of the a-helix, much like rotation of a ball-and-socket joint.…”

“…The orientations differ by rotation of the nucleotide about its 5'-phosphate in the fiist turn of the a-helix, much like rotation of a ball-and-socket joint. The structures with the joint rotated so that ribose is closest to the PRPP loop also include Mg2+ (Tomchick et al, 1994;Schumacher et al, 1996;Chen et al, 1997). In these structures, Mg2+ is coordinated by the ribose 2'-OH and 3'-OH and by the two acidic side chains that are a hallmark of PRPP loop sequences (Asp 366-Asp 367 of E. coli GPATase).…”

“…Nucleotide specificity at these sites differs in the E. coli and B. subtilis enzymes. AMP binds preferentially to the C site and GMP to the A site of E. coli GPATase (Zhou et al, 1994), whereas GMP is preferred in the B. subtilis C site and ADP in the A site (Chen et al, 1997).…”

Discoveries in Bacterial Nucleotide Metabolism

“…Like a number of biosynthetic enzymes leading to multiple end products, PRPP amidotransferase is subject to synergistic allosteric inhibition; the end products AMP and GMP (or GDP) are much more effective inhibitors in combination than they are individually (56). Through a combination of x-ray crystallography and characterization of mutant enzymes constructed in vitro, it has been possible to map the sites of nucleotide binding to B. subtilis PRPP amidotransferase quite precisely and to identify the interactions between allosteric and catalytic sites that account for synergistic inhibition (57). I am not aware of any other instance of synergistic inhibition for which this has been accomplished.…”