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.
Single tryptophan residues were incorporated into each of three peptide segments that play key roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substrate binding were used to calculate binding constants and to detect the structural rearrangements that coordinate reactions at active sites for glutamine hydrolysis and PRTase catalysis. Pre-steady state kinetics of enzyme.PRPP and enzyme.PRPP.glutamine complex formation were determined from stopped-flow fluorescence measurements. The kinetics of the formation of the enzyme.PRPP complex were consistent with a model with two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzyme isomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely the rate-limiting step in the reaction of PRPP with NH(3). The pre-steady state kinetics for binding glutamine to the binary enzyme. PRPP complex could also be fit to a model involving rapid equilibrium binding of glutamine followed by an enzyme isomerization step. The changes monitored by fluorescence account for the interconversions between "end state" structures determined previously by X-ray crystallography and define an intermediate enzyme.PRPP conformer.
The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Glutamine PRPP 1 amidotransferase, an N-terminal nucleophile type glutamine amidotransferase (1), catalyzes the first step in purine nucleotide synthesis, shown by Equation 1. This reaction takes place in two steps (Equations 2 and 3).X-ray structures have defined the structure-function relationship (2, 3). An N-terminal glutaminase domain catalyzes the first half-reaction (Equation 2) and a C-terminal PRTase domain the second step (Equation 3). These catalytic sites are separated by 16 Å. Binding of PRPP to the PRTase catalytic site activates the glutaminase step (4, 5), a feature that prevents the wasteful hydrolysis of glutamine independent of PRA synthesis. We have investigated two key questions regarding the enzyme mechanism. First, how is the PRPP-binding signal communicated to the glutamine site over a distance of 16 Å? Second, how is NH 3 produced at the glutamine site sequestered from solvent and delivered to the PRTase domain for nucleophilic attack on PRPP to produce PRA and PP i ? The substrate for the second half-reaction, shown by Equation 3, is NH 3 , not NH 4 ϩ (4). X-ray crystal structures of an Escherichia coli ligandfree enzyme (2) and an enzyme-substrate analog ternary complex (3) have provided the framework to investigate these questions. The ligand-free and ternary complex structures have been referred to as state I and state III conformers, respectively (6). The structure of the state I enzyme is incompatible with catalysis. The unfavorable properties include: (i) the PRPP site is exposed to solvent; (ii) an important arginine residue (Arg 73 ) needed for glutamine binding is unfavorably positioned; and (iii) sites for glutamine hydrolysis and reaction of NH 3 with PRPP are separated by a 16 Å solvent-exposed space. These barriers to catalysis are corrected in the structure of the state III enzyme-substrate ternary complex. A PRTase "flexible loop" (residues 326 -350) has closed over the bound PRPP, thus protecting it from hydrolysis. Arg 73 is optimally positioned for binding glutamine and the glutamine, and PRTase sites are connected by a 20 Å NH 3 channel. However, the x-ray structures of the two enzyme conformers do not indicate how glutamine binds because in each conformer the glutamine site is closed thus restricting entry to the site. Thus, glutamine must initially bind to a different enzyme conformer. Recently, we have engineered enzymes containing a single tryptophan fluorescence reporter in positions that change conformation upon formation of the enzyme-substrate ternary complex (6). Measurements of steady state and pre-steady state intrinsic tryptophan fluorescence have identified an intermediate state II enzyme⅐PRPP conformer. ...
GPAT and AIRC encode enzymes for steps one and six plus seven respectively in the pathway for de novo purine nucleotide synthesis in vertebrates. The human GPAT and AIRC genes are divergently transcribed from a 558 bp intergenic promoter region. Cis-acting sites and transcription factors important for bidirectional expression were identified. A cluster of sites between nt 215 and 260 are essential, although not sufficient, for expression of both genes. Two proteins from HepG2 cell nuclear extract, identified as NRF-1 and Sp1, bound to the promoter at sites within the 215-260 region. NRF-1 was required for stable binding of Sp1. Deletion of a 5'promoter region including nt 215-260 resulted in decreased expression of GPAT and AIRC in transfected HepG2 cells. The decreased expression was accounted for by point mutations in an NRF-1 site and either of two flanking sites for Sp1. These transcription factors account in part for the coordinated expression of human GPAT and AIRC.
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