Thermodynamics of aminoglycoside binding to aminoglycoside-3'-phosphotransferase IIIa studied by isothermal titration calorimetry.
The aminoglycoside-3'-phosphotransferase IIIa [APH(3')-IIIa] phosphorylates aminoglycoside antibiotics and renders them ineffective against bacteria. APH(3')-IIIa is the most promiscuous aminoglycoside phosphotransferase enzyme, and it modifies more than 10 different aminoglycoside antibiotics. A wealth of information exists about the enzyme; however, thermodynamic properties of enzyme-aminoglycoside complexes are still not known. This study describes the determination of the thermodynamic parameters of the binary enzyme-aminoglycoside and the ternary enzyme-metal-ATP-aminoglycoside complexes of structurally related aminoglycosides using isothermal titration calorimetry. Formation of the binary enzyme-aminoglycoside complexes is enthalpically driven and exhibits a strongly disfavored entropic contribution. Formation of the ternary enzyme-metal-ATP-aminoglycoside complexes yields much smaller negative DeltaH values and more favorable entropic contributions. The presence of metal-ATP generally increases the affinity of aminoglycosides to the enzyme. This is consistent with the kinetic mechanism of the enzyme in which ordered binding of substrates occurs. However, the observed DeltaH values neither correlate with kinetic parameters k(cat), K(m), and k(cat)/K(m) nor correlate with the molecular size of the substrates. Comparison of the thermodynamic properties of the complexes formed by structurally similar aminoglycosides indicated that the 2'- and the 6'-amino groups of the substrates are involved in binding to the enzyme. Thermodynamic properties of the complexes formed by aminoglycosides differing only at the 3'-hydroxyl group suggested that the absence of this group does not alter the thermodynamic parameters of the ternary APH(3')-IIIa-metal-ATP-aminoglycoside complex. Our results also indicate that protonation of ligand and protein ionizable groups is coupled to the complex formation between aminoglycosides and APH(3')-IIIa. Comparison of DeltaH values for different aminoglycoside-enzyme complexes indicates that enzyme and substrates undergo significant conformational changes in complex formation.
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Graduate School of Genome Science and Technology, Department of Biochemistry and Cellular and Molecular Biology, and Center of Excellence in Structural Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
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D000255:Adenosine Triphosphate / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000617:Aminoglycosides / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002151:Calorimetry / Q000379:methods; D019277:Entropy; D006595:Hexosamines / Q000737:chemistry; D006878:Hydroxides / Q000737:chemistry; D007612:Kanamycin / Q000031:analogs & derivatives* / Q000737:chemistry; D019868:Kanamycin Kinase / Q000737:chemistry*; D046911:Macromolecular Substances / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics
D000588:Amines; D000617:Aminoglycosides; D006595:Hexosamines; D006878:Hydroxides; D046911:Macromolecular Substances; C001495:bekanamycin; D007612:Kanamycin; D000255:Adenosine Triphosphate; C031356:hydroxide ion; D019868:Kanamycin Kinase; C007265:2-deoxystreptamine; D002118:Calcium