Recent structural studies reveal that protein conformational transitions are fundamental to signaling, enzyme catalysis, and assembly of cellular structures. Understanding how the interconversion between different folded structures affects function is challenging but would create a huge impact in treating a large number of diseases that are linked to signaling cascades or enzymes. Although advanced techniques in structural biology have been well-developed to decipher the effects of changes in structures of biological molecules into their functions, these methods have been applied mostly to low molecular weight systems. Enzymes, however, are typically large oligomeric proteins with complex molecular features, and their function is often regulated by long-range communication between structural domains mediated by substrate binding. Therefore, there is a critical need to increase our understanding of how modulation of the local conformational dynamics upon ligand binding propagates into broader scale inter-domain rearrangements that ultimately determine the function of complex multi-domain proteins. Enzyme I (EI) serves as an interesting model for investigating the interplay between regional dynamics and their dissipating effects. During my research project, I have developed an NMR-based enzymatic assay to investigate the contribution of the EI monomer-dimer equilibrium in the regulation of its enzymatic activity. In addition, the same method was used to study how the EI oligomerization equilibrium determines pluripotency of the small molecule metabolite α-ketoglutarate (αKG) against the enzyme. In follow-up work, I have investigated the structure and dynamics of the elusive monomeric state of EI. Indeed, although the dimeric state of EI has been deeply characterized for structure, dynamics, and function, the monomeric state of EI is difficult to observe at the experimental conditions commonly used in biophysical approaches. Using a combination of protein engineering and pressure perturbation, I was able to isolate the monomeric state of EI and perform a comprehensive structural and functional characterization of the enzyme by NMR. My study unveils that the catalytic loops near the dimer interface become disordered upon monomerization and, therefore, fail to bind the substrate in the active site. These data explain why only dimeric EI is active and required for a fully functional phosphotransferase system (PTS). Finally, I have explored the possibility of inhibiting bacterial EI with small organic molecules. PTS is ubiquitous and indispensable in prokaryotes but is absent in eukaryotes. Therefore, blocking the PTS pathway is a possible strategy for the development of new antimicrobial drugs. EI is the first enzyme in PTS and is highly conserved in bacteria. Thus, EI is the ideal target for a screening campaign aimed at inhibiting the PTS pathway. Here, I have used NMR-based fragment screening to identify novel inhibitors of EI. I have found three molecular fragments that allosterically inhibit the phosphoryl transfer reaction catalyzed by EI by interacting with the enzyme at a surface pocket located more than 10 Å away from the active site. My study provides the basis for developing second-generation allosteric inhibitors of EI that can potentially address the antibiotic-resistant problem.