Kinetic Studies of Aminoglycoside Acetyltransferase and Phosphotransferase from Staphylococcus aureus RPAL

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Abstract
In the S. aureus strain harboring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from 2 inactivating reactions catalyzed by a 6''-N-aminoglycoside acetyltransferase and a 2"-O-aminoglycoside phosphotransferase. These enzymes are copurified with a constant ratio between the 2 activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity were determinated from studies of initial velocities and product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of 1 reaction were tested as effectors of the other reaction. No interaction between the 2 activities was observed. The GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltranferase against thermal inactivation, which suggests that the 2 activities may be associated.