Tryptophan Fluorescence Monitors Multiple Conformational Changes Required for Glutamine Phosphoribosylpyrophosphate Amidotransferase Interdomain Signaling and Catalysis^,

Abstract

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.

Interdomain Signaling in Glutamine Phosphoribosylpyrophosphate Amidotransferase

“…Enzyme Production and Purification-Wild type E. coli glutamine PRPP amidotransferase was produced from plasmid pETpurF (6). Mutations were constructed by the method of Kunkel et al (9) using pETpurF phagemid DNA.…”

“…Here we report the construction and analyses of mutant enzymes to evaluate the structure-based mechanism for interdomain signaling. New tryptophan reporters in the glutaminase domain detect the interdomain signaling upon PRPP binding that was not detected previously (6).…”

“…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.…”

“…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.…”

Gain of Glutaminase Function in Mutants of the Ammonia-specific Frog Carbamoyl Phosphate Synthetase

“…It is not yet clear how extensive the underlying changes are (relative to a functional tunnel), whether they are confined to one or both domains, or what path is taken by ammonia between the GAT and SYN active sites. The molecular basis for coordination of the GAT and SYN active sites connected by a channel has been elucidated for two other GATs, glutamine phosphoribosylpyrophosphate amidotransferase (24,25) and imidazole glycerol phosphate synthase (26 -28), and is based on a cycle of conformational changes that control access of substrates, intermediates, and bulk solvent to the active sites and/or the tunnel. Thus far, data for CPS are limited to a single solved conformation of eCPS and identification of 10 GAT residues that appear to line the interior of the tunnel (20).…”