In this work, the binding of coenzyme A (CoASH) to the aminoglycoside acetyltransferase (3)-IIIb (AAC) is studied by several experimental techniques. These data represent the first thermodynamic and kinetic characterization of interaction of a cofactor with an enzyme that modifies the 2-deoxystreptamine ring (2-DOS) common to all aminoglycoside antibiotics. Acetyl coenzyme A (AcCoA) was the preferred substrate, but propionyl and malonyl CoA were also substrates. CoASH associates with two different sites on AAC as confirmed by ITC, NMR, and fluorescence experiments: one with a high-affinity, catalytic site and a secondary, low-affinity site that overlaps with the antibiotic binding pocket. The binding of CoASH to the high-affinity site occurs with a small, unfavorable enthalpy and a favorable entropy. Binding to the second site is highly exothermic and is accompanied by an unfavorable entropic contribution. The presence of an aminoglycoside alters the binding of CoASH to AAC dramatically such that the binding occurs with a favorable enthalpy (DeltaH < 0) and an unfavorable entropy (TDeltaS < 0). This is irrespective of which aminoglycoside is the cosubstrate and occurs without a significant change in the affinity of CoASH for AAC. Also, antibiotics eliminate binding of CoASH to the second site. These data allowed the enthalpies of all six equilibria present in a ternary system (AAC-antibiotic-coenzyme) to be determined for the first time for an aminoglycoside-modifying enzyme. NMR experiments also shed light on the dynamic nature of AAC as fast, slow, and intermediary exchanges between apoenzyme- and coenzyme-bound forms were observed.