The aminoglycoside (AG) 3'-phosphotransferases [APH(3')s] are an important class of modifying enzymes which confer high-level resistance to those AGs actively modified by the enzymes. They catalyze the transfer of the terminal phosphate from ATP to the drug, thus preventing the AG s action at the 70S ribosome. These enzymes, which utilize ATP as a co-substrate, appear from amino acid alignments to be part of a much larger superfamily of kinases and ATP-binding proteins. Structure-function analyses have been initiated in our laboratory for APH(3')-II, whose gene was derived from transposon Tn5. Site-directed mutagenesis of the cloned APH(3')-II gene was used to genetically examine the residues in two highly-conserved motifs proposed to participate in ATP binding. Several of these residues, in fact, were shown to affect the enzyme s affinity for ATP. We have also initiated studies using photoaffinity labelling of APH(3')-II with the ATP analogs, 8-azido-ATP and 2-azido-ATP. We have shown that 8-N₃ATP and 2-N₃ATP can be substituted for ATP in the APH(3')-II catalyzed phosphorylation of kanamycin; such findings indicate that the interaction of these photoaffinity analogs of ATP with APH(3')-II is biologically relevant. One of the best-characterized of the APH(3') enzymes is APH(3')-IIIa, the first of the group whose structure has been analyzed by x- ray crystallography. Several studies have demonstrated that this enzyme functions by a Theorell-Chance mechanism. Moreover, the architecture of the enzyme, crystallized in the presence of ADP has revealed residues in the ATP-binding pocket which are likely to play important roles in catalysis. Once the results from biochemical analyses can be correlated with those from mutagenesis studies and x-ray crystallography, a clearer picture of the active site will be provided for an important class of AG-modifying enzymes and phosphotransferases. This picture will also allow a better understanding of these enzymes within the greater context of kinases and nucleotide-binding proteins.