Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase

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 act ivate the reaction at the N-terminal glutaminase domain. Interdomain signal ing was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in th e glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Tr p(482) in the C-terminal helix (residues 471-492) reported fluorescence cha nges in the glutaminase domain upon binding of PRPP and glutamine. The fluo rescence changes were perturbed by Ile(335) and Tyr(74) mutations that disr upt interdomain signaling. Fluoresence titrations of PRPP and glutamine bin ding indicated that signaling defects increased the K-d for glutamine but h ad little or no effect on PRPP binding. It was concluded that the contact b etween Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling . Analysis of enzymes with mutations in the glutaminase domain C-terminal h elix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.