Akademik Veri Yönetim Sistemi
BIOCHEMISTRY, cilt.47, sa.1, ss.40-49, 2008 (SCI-Expanded)
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 Delta H in H2O relative to D2O (Delta Delta H(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative Delta C-p values were observed for binding of aminoglycosides to APH. Delta C-p for the APH-neomycin complex was -1.6 kcal(.)mol(-1.)deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding Delta C-p values of -0.7 kcal(.)mol(-1.)deg(-1) and -3.8 kcal(.)mol(-1.)deg(-1) below and above 30 degrees C, respectively. Neither the change in cessible surface area (Delta ASA) nor contributions from heats of ionization were sufficient to explain the large negative Delta C-p values. Most significantly, N-15-H-1 HSQC experiments showed that temperature-dependent shifts of the backkbone 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 degrees C and 38 degrees 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 Delta C-p 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.