Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis

Single tryptophan residues were incorporated into each of three peptide seg ments that play key roles in the structural transition of ligand-free, inac tive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the a ctive enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluor escence quenching were used to monitor changes in a phosphoribosyltransfera se (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. St eady 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 PRTa se catalysis. Pre-steady state kinetics of enzyme PRPP and enzyme PRPP glut amine complex formation were determined from stopped-now fluorescence measu rements. The kinetics of the formation of the enzyme PRPP complex were cons istent with a model with two or more steps in which rapid equilibrium bindi ng of PRPP is followed by a slow enzyme isomerization. This isomerization i s ascribed to the closing of the PRTase flexible loop and is likely the rat e-limiting step in the reaction of PRPP with NH3. The pre-steady state kine tics 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 accoun t for the interconversions between "end state" structures determined previo usly by X-ray crystallography and define an intermediate enzyme PRPP confor mer.