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

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• 作者：Sihong Chen ; John W. Burgner ; Joseph M. Krahn ; Janet L. Smith ; and Howard Zalkin
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：September 7, 1999
• 年：1999
• 卷：38
• 期：36
• 页码：11659 - 11669
• 全文大小：389K
• 年卷期：v.38,no.36(September 7, 1999)
• ISSN：1520-4995

文摘

Single tryptophan residues were incorporated into each of three peptide segments that playkey roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP)amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence andfluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexibleloop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substratebinding were used to calculate binding constants and to detect the structural rearrangements that coordinatereactions 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 fluorescencemeasurements. The kinetics of the formation of the enzyme·PRPP complex were consistent with a modelwith two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzymeisomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely therate-limiting step in the reaction of PRPP with NH₃. The pre-steady state kinetics for binding glutamineto the binary enzyme·PRPP complex could also be fit to a model involving rapid equilibrium binding ofglutamine followed by an enzyme isomerization step. The changes monitored by fluorescence accountfor the interconversions between "end state" structures determined previously by X-ray crystallographyand define an intermediate enzyme·PRPP conformer.