Dynamic processes are implicit in the catalytic function of all enzymes. Although there is considerable evidence both from theory and experiment that many enzymes are inherently flexible, the fundamental question of how, or even if, protein fluctuations couple to catalytic functions remains unanswered. These issues will be addressed using state- of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well-characterized enzyme, dihydrofolate reductase from E. coli, in all of the intermediate states implicated along its reaction pathway. DHFR is the target enzyme for anti-folate drugs such as the anti- cancer agent methotrexate and the anti-bacterial trimethoprim and is of considerable medical significance. The proposed research will provide direct experimental data on the intrinsic molecular dynamics of this important enzyme and insights into the mechanism by which conformational fluctuations are coupled to interactions with substrate, cofactor, and products in the key intermediates of the catalytic cycle. Dynamic """"""""hot spots"""""""" identified through the NMR relaxation studies will be targeted for mutagenesis (Project 3). The insights into DHFR dynamics resulting from this research will provide a basis for the theoretical studies of Project 4.
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