Abstract
This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3‘)-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative ΔH in H2O relative to D2O (ΔΔH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative ΔCp values were observed for binding of aminoglycosides to APH. ΔCp for the APH−neomycin complex was −1.6 kcal·mol-1·deg-1. A break at 30 °C was observed in the APH−kanamycin complex yielding ΔCp values of −0.7 kcal·mol-1·deg-1 and −3.8 kcal·mol-1·deg-1 below and above 30 °C, respectively. Neither the change in accessible surface area (ΔASA) nor contributions from heats of ionization were sufficient to explain the large negative ΔCp values. Most significantly, 15N−1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 °C only in the APH−kanamycin complex in spectra collected between 21 °C and 38 °C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect ΔCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.