In this work, for the first time, we report pKa values of the amino functions in a target-bound aminoglycoside antibiotic, which permitted dissection of the thermodynamic properties of an enzyme-aminoglycoside complex. Uniformly enriched 15N-neomycin was isolated from cultures of Streptomyces fradiae and used to study its binding to the aminoglycoside phosphotransferase(3')-IIIa (APH) by 15N NMR spectroscopy. 15N NMR studies showed that binding of neomycin to APH causes upshifts of approximately 1 pKa unit for the amines N2' and N2' '' while N6' experienced a 0.3 pKa unit shift. The pKa of N6' '' remained unaltered, and resonances of N1 and N3 showed significant broadening upon binding to the enzyme. The binding-linked protonation and pH dependence of the association constant (Kb) for the enzyme-aminoglycoside complex was determined by isothermal titration calorimetry. The enthalpy of binding became more favorable (negative) with increasing pH. At high pH, binding-linked protonation was attributable mostly to the amino functions of neomycin; however, at neutral pH, functional groups of the enzyme, possibly remote from the active site, also underwent protonation/deprotonation upon formation of the binary enzyme-neomycin complex. The Kb for the enzyme-neomycin complex showed a complicated dependence on pH, indicating that multiple interactions may affect the affinity of the ligand to the enzyme and altered conditions, such as pH, may favor one or another. This work highlights the importance of determining thermodynamic parameters of aminoglycoside-target interactions under different conditions before making attributions to specific sites and their effects on these global parameters.