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The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.