Aminoglycoside-modifying enzymes are responsible for high-level bacterial resistance to the aminoglycoside antiobiotics and compromise the effectiveness of these drugs for treating serious infections. It is likely that via mutational alteration the genes for these enzymes provide a reservoir from which bacterial resistance to newly designed aminoglycosides might emerge. This study proposes experiments aimed at characterizing the active site for an aminoglycoside 3'-phosphotransferase [APH(3')-II]. This enzyme normally confers bacterial resistance to kanamycin, but not to its semi-synthetic derivative, amikacin. This is true even though the enzyme modifies amikacin, albeit with much less catalytic efficiency than for kanamycin. Further kinetic studies of the purified APH(3')-II will be undertaken to identify potential inhibitors. Additionally, available sequence data has been examined and a number of conserved amino acids between several APH(3') enzymes have been identified. Modifying reagents specific for certain residues have been used to treat APH(3')-II to determine which of these are necessary for function. Thusfar histidine has been implicated in binding the AG, while tyrosine seems primarily to bind the ATP. The ATP-binding site will be further probed specifically using photoaffinity analogs of ATP. Together with site-directed mutagenesis of the cloned APH(3')-II gene, these approaches should provide us with insight as to the structure and function of this class of enzymes. Perhaps this will aid in determining the genetic potential of such enzymes to improve their recognition of new substrates, such as amikacin.