Transient kinetics of aminoglycoside phosphotransferase(3′)-IIIa reveals a potential drug target in the antibiotic resistance mechanism

► APH(3′)-IIIa is a bacterial kinase responsible for antibiotics resistance. ► Release of ADP is the rate limiting step of Kanamycin A phosphorylation. ► An ADP-enzyme complex is the main steady state intermediate. ► A strategy to prevent the phosphorylation of aminoglycosides is uncompetitive inhibition. ► A good target for uncompetitive inhibitors is the ADP-enzyme complex.