This thesis examines ligand binding and associated conformational dynamics of the flavoenzyme, UDP-galactopyranose mutase (UGM), using steady-state fluorescence of its tryptophan residues and flavin adenine dinucleotide (FAD) cofactor. The work is supported by photochemical characterization of the unbound enzyme. Substrate binding triggers protein conformational changes sensed by W70/W290, but not the strictly-conserved binding pocket W160. The changes are proposed to involve substrate-directed closure of a “recognition loop”, through coordinated interaction with the distal FAD and W160 sites. This model is validated by compromising interactions at the FAD site, through oxidation or binding of the non-bridging ligand, UDP, and at the W160 site, through mutagenesis, and by molecular dynamics simulations and STD-NMR experiments of collaborators. The mechanistic insight is directing design of molecular probes and potential inhibitors of UGM, and the conclusions regarding the role of protein dynamics in enzyme specificity are expected to be general.
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