The thermodynamic and kinetic properties of interactions of antibiotics with the aminoglycoside acetyltransferase (3)-IIIb (AAC) are determined with several experimental methods. These data represent the first such characterization of an enzyme that modifies the 2-deoxystreptamine ring common to all aminoglycoside antibiotics. Antibiotic substrates for AAC include kanamycin A, kanamycin B, tobramycin, sisomicin, neomycin B, paromomycin, lividomycin A, and ribostamycin. Kinetic studies show that kanamycin group aminoglycosides have higher k(cat) values than members of the neomycin group. Only small aminoglycosides without intraring constraints show substrate inhibition. Isothermal titration calorimetry (ITC) and fluorescence measurements are consistent with a molecular size-dependent stoichiometry where binding stoichiometries are 1.5-2.0 for small antibiotics and 1.0 for larger. Antibiotic-enzyme interaction occurs with a favorable enthalpy (DeltaH < 0) and a compensating unfavorable entropy (TDeltaS < 0). The presence of coenzyme A significantly increases the affinity of the antibiotic for AAC. However, the thermodynamic properties of its ternary complexes distinguish this enzyme from other aminoglycoside-modifying enzymes (AGMEs). Unlike other AGMEs, the enthalpy of binding becomes more favored by 1.7-10.0-fold in the presence of the cosubstrate CoASH, while the entropy becomes 2.0-22.5-fold less favored. The overall free energy change is still only 1.0-1.9 kcal/mol from binary to ternary for all antibiotics tested, which is similar to those for other aminoglycoside-modifying enzymes. A computationally derived homology model provides structural support for these conclusions and further indicates that AAC is likely a member of the GCN5-related acetyltransferase family of proteins.
In this work, the binding of coenzyme A (CoASH) to the aminoglycoside acetyltransferase (3)-IIIb (AAC) is studied by several experimental techniques. These data represent the first thermodynamic and kinetic characterization of interaction of a cofactor with an enzyme that modifies the 2-deoxystreptamine ring (2-DOS) common to all aminoglycoside antibiotics. Acetyl coenzyme A (AcCoA) was the preferred substrate, but propionyl and malonyl CoA were also substrates. CoASH associates with two different sites on AAC as confirmed by ITC, NMR, and fluorescence experiments: one with a high-affinity, catalytic site and a secondary, low-affinity site that overlaps with the antibiotic binding pocket. The binding of CoASH to the high-affinity site occurs with a small, unfavorable enthalpy and a favorable entropy. Binding to the second site is highly exothermic and is accompanied by an unfavorable entropic contribution. The presence of an aminoglycoside alters the binding of CoASH to AAC dramatically such that the binding occurs with a favorable enthalpy (DeltaH < 0) and an unfavorable entropy (TDeltaS < 0). This is irrespective of which aminoglycoside is the cosubstrate and occurs without a significant change in the affinity of CoASH for AAC. Also, antibiotics eliminate binding of CoASH to the second site. These data allowed the enthalpies of all six equilibria present in a ternary system (AAC-antibiotic-coenzyme) to be determined for the first time for an aminoglycoside-modifying enzyme. NMR experiments also shed light on the dynamic nature of AAC as fast, slow, and intermediary exchanges between apoenzyme- and coenzyme-bound forms were observed.
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 DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp 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 degrees 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 DeltaCp 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.
In this work, hydrogen-deuterium exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3′-IIIa (APH), a protein of intense interest due to its involvement in conferring antibiotic resistance to both Gram negative and Gram positive microorganisms. This represents the first characterization of dynamic properties of an aminoglycosidemodifying enzyme. Herein we describe in vitro dynamics of apo, binary, and ternary complexes of APH with kanamycin A, neomycin B, and metal-nucleotide. Regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 h of exposure to D 2 O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange, and ∼30% of the amides remain unexchanged up to 95 h after exposure to D 2 O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to the enzyme-aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby β-sheet, thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide a molecular basis for broad substrate selectivity of APH.
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