Thermodynamic Studies of Aminoglycoside Phosphotransferase (3')-III with Substrates

Aminoglycoside-3'-phosphotransferase IIIa [APH (3')] is one of the most important aminoglycoside modifying enzyme which inactivates a broad spectrum of aminoglycosides by Metal ATP-dependent phosphorylation. In this work, the affinity and kinetic properties of the aminoglycoside phosphotransferase (3')-III, with its substrates depending on metal ions (Magnesium vs. Manganese), were systematically determined by kinetic assay, electron paramagnetic resonance (EPR) and fluorescence spectra. EPR studies showed metal ions binding to enzymes behaved in different manner with and without nucleotides. The stoichiometry and dissociation constants of Mg binding to the enzyme complex were determined by the competition between Mg and Mn. The affinity of AMPPCP increased in the presence metal ions in the fluorescence studies, even after addition of antibiotics. These studies provide more evidences for substrate specificity of aminoglycoside enzymes depending on metal ligands. The global view on the affinity for all substrates binding to APH is currently available for the understanding of mechanism for bacterial resistance.