Résumé
► 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.
Aminoglycoside phosphotransferases are bacterial enzymes responsible for the inactivation of aminoglycoside antibiotics by O-phosphorylation. It is important to understand the mechanism of enzymes in order to find efficient drugs. Using rapid-mixing methods, we studied the transient kinetics of aminoglycoside phosphotransferase(3′)-IIIa. We show that an ADP-enzyme complex is the main steady state intermediate. This intermediate interacts strongly with kanamycin A to form an abortive complex that traps the enzyme in an inactive state. A good strategy to prevent the inactivation of aminoglycosides would be to develop uncompetitive inhibitors that interact with this key ADP-enzyme complex.