Enzyme−Substrate Interactions with an Antibiotic Resistance Enzyme: Aminoglycoside Nucleotidyltransferase(2‘ ‘)-Ia Characterized by Kinetic and Thermodynamic Methods

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Abstract
Aminoglycoside nucleotidyltransferase(2‘ ‘)-Ia is one of the most often detected enzymes in aminoglycoside-resistant bacteria. Despite its prevalence, little biochemical and biophysical work has been reported for this enzyme. In the current study, substrate specificity and temperature dependence of kcat are determined by kinetic assays. Dissociation constants and thermodynamic properties of enzyme−substrate complexes are determined by isothermal titration calorimetry, electron paramagnetic resonance, and fluorescence spectroscopy. Kinetic studies show that aminoglycosides with 2‘-NH2 are better substrates (higher kcat/Km) than ones with 2‘-OH when magnesium(II) is used as the catalytically required divalent cation. The activity is reduced 10-fold for substrates with 2‘-NH2 when manganese(II) replaces magnesium as the required metal. However, kanamycin A, which has a 2‘-OH, shows a much smaller decrease in activity when manganese substitutes for magnesium as the divalent cation. Temperature dependence studies show the activation energy of catalysis to be 19.2 kcal/mol and the temperature optimum between 30 and 32 °C. The binding of the aminoglycoside substrate tobramycin to the enzyme occurs with a favorable enthalpy which compensates for a large entropic penalty to yield a negative ΔG value for the complex formation. Enthalpy of binding is less exothermic in the presence of metal−nucleotide. However, due to the more favorable entropy, a more favorable ΔG is observed for the formation of the enzyme−metal−nucleotide:aminoglycoside complex. Tobramycin binds to ANT(2‘ ‘) with a dissociation constant of 0.6 μM, which is further reduced by 3-fold when metal−nucleotide is present. Binding of ATP to the enzyme is determined to be very weak in the absence of a divalent cation, and becomes 2 orders of magnitude tighter when magnesium or manganese is present. Binding studies also show that, in addition to binding to the enzyme in the form of metal−nucleotide complex, a second catalytically required metal binds to an additional site on the enzyme.