Aminoglycoside nucleotidyltransferase(2″)-Ia (ANT) catalyzes the covalent modification of certain aminoglycoside antibiotics and imparts resistance to bacteria which possess this enzyme. Isothermal titration calorimetry (ITC) experiments using two structurally similar substrates (kanamycin A and kanamycin B) and a structurally different inhibitor (neomycin) were performed to determine pH and proton linkage effects on thermodynamic properties of enzyme–aminoglycoside complexes. Data showed that there was a decrease in pKa for one or more carboxyl groups on the enzyme that interacts with 2′-amino group of neomycin or kanamycin B. Protonation state of this site exerted a significant effect on the binding affinity of ligand to ANT. ITC experiments also showed that the loss of affinity at higher pH values was not as pronounced for neomycin compared to kanamycins. This difference is a result of higher pK a s for free neomycin which are up shifted in the ANT–neomycin complex. The decrease in affinity and differences in binding linked protonation above and below the pH optimum cannot be fully explained by these two factors. Therefore, the formation of ANT–aminoglycoside complexes involves protonation and deprotonation of multiple functional groups on both ligand and enzyme which also contribute to the pH profile of the complex formation.