Kinetic Mechanism of Enterococcal Aminoglycoside Phosphotransferase 2' '-Ib

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• 作者：Marta Toth ; Jaroslav Zajicek ; Choonkeun Kim ; Joseph W. Chow ; Clyde Smith ; Shahriar Mobashery ; Sergei Vakulenko
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：May 8, 2007
• 年：2007
• 卷：46
• 期：18
• 页码：5570 - 5578
• 全文大小：97K
• 年卷期：v.46,no.18(May 8, 2007)
• ISSN：1520-4995

文摘

The major mechanism of resistance to aminoglycosides in clinical bacterial isolates is thecovalent modification of these antibiotics by enzymes produced by the bacteria. Aminoglycoside 2' '-Ibphosphotransferase [APH(2' ')-Ib] produces resistance to several clinically important aminoglycosides inboth Gram-positive and Gram-negative bacteria. Nuclear magnetic resonance analysis of the product ofkanamycin A phosphorylation revealed that modification occurs at the 2' '-hydroxyl of the aminoglycoside.APH(2' ')-Ib phosphorylates 4,6-disubstituted aminoglycosides with k_cat/K_m values of 10⁵-10⁷ M^-1 s^-1_,while 4,5-disubstituted antibiotics are not substrates for the enzyme. Initial velocity studies demonstratethat APH(2' ')-Ib operates by a sequential mechanism. Product and dead-end inhibition patterns indicatethat binding of aminoglycoside antibiotic and ATP occurs in a random manner. These data, together withthe results of solvent isotope and viscosity effect studies, demonstrate that APH(2' ')-Ib follows the randomBi-Bi kinetic mechanism and substrate binding and/or product release could limit the rate of reaction.