Thermodynamic Characterization of Aminoglycoside-3′-Phosphotransferase IIIa

Aminoglycoside-3′-Phosphotransferase IIIa is a widespread, promiscuous member of the phosphotransferase family of aminoglycoside modifying enzymes. This study provides results of combined calorimetry/NMR experiments to characterize and dissect the global thermodynamic properties of aminoglycoside–APH(3′)-IIIa complexes. Aminoglycoside binding to APH(3′)-IIIa is enthalpically driven with strong entropic penalty. 2′- and 6′-amino groups have significant contributions to the observed binding parameters. Formation of APH(3′)-IIIa complexes with substrate aminoglycosides shows a complex dependence on pH and is linked to protonation and deprotonation of both ligand and enzyme groups. We report pK_a upshifts of ~1 unit for N2′ and N2′′′ groups of enzyme-bound neomycin B while the pK_a of N6′ changes by 0.3 unit and N6′′′ experiences no shift. Isotopic solvent and heat capacity change studies strongly suggest differential effects and reorganization of solvent in kanamycin and neomycin class complexes of the enzyme. We also determined unusually high binding ΔCp values in the range of -0.7 to -3.8 kcal/mol·deg which were not explained by changes in the solvent accessible surface area. A break at 30°C was observed in the ΔCp plot and temperaturedependent backbone amide proton chemical shifts of four residues surrounding the binding site of kanamycin-APH(3′)-IIIa complex. These results may indicate specific solvent reorganization sites away from the binding site of the enzyme.