Kinetic mechanism of enterococcal aminoglycoside phosphotransferase 2"-Ib.
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.
Toth
Marta
M
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Zajicek
Jaroslav
J
Kim
Choonkeun
C
Chow
Joseph W
JW
Smith
Clyde
C
Mobashery
Shahriar
S
Vakulenko
Sergei
S
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D024881:Drug Resistance, Bacterial; D016984:Enterococcus faecium / Q000201:enzymology*; D007612:Kanamycin / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010865:Pilot Projects; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007612:Kanamycin; D017853:Phosphotransferases (Alcohol Group Acceptor); C415966:aph(2')-Ib protein, Enterococcus faecium