Abstract
The major mechanism of resistance to aminoglycosides in clinical bacterial isolates is the covalent modification of these antibiotics by enzymes produced by the bacteria. Aminoglycoside 2''-Ib phosphotransferase [APH(2'')-Ib] produces resistance to several clinically important aminoglycosides in both Gram-positive and Gram-negative bacteria. Nuclear magnetic resonance analysis of the product of kanamycin A phosphorylation revealed that modification occurs at the 2''-hydroxyl of the aminoglycoside. APH(2'')-Ib phosphorylates 4,6-disubstituted aminoglycosides with kcat/Km values of 10(5)-10(7) M-1 s-1, while 4,5-disubstituted antibiotics are not substrates for the enzyme. Initial velocity studies demonstrate that APH(2'')-Ib operates by a sequential mechanism. Product and dead-end inhibition patterns indicate that binding of aminoglycoside antibiotic and ATP occurs in a random manner. These data, together with the results of solvent isotope and viscosity effect studies, demonstrate that APH(2'')-Ib follows the random Bi-Bi kinetic mechanism and substrate binding and/or product release could limit the rate of reaction.
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