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The Kinetic Mechanism of AAC(3)-IV Aminoglycoside Acetyltransferase from Escherichia coli

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Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461
Cite this: Biochemistry 2005, 44, 49, 16275–16283
Publication Date (Web):November 16, 2005
https://doi.org/10.1021/bi051777d
Copyright © 2005 American Chemical Society

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    Abstract

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    The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.

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     This work was supported by NIH Grant AI60899.

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     Corresponding author. Tel:  (718) 430-3096. Fax:  (718) 430-8565. E-mail:  [email protected].

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    Double reciprocal plot of initial rate data at varying ribostamycin and fixed AcCoA concentrations (Figure S1), dead-end inhibition patterns (Figure S2), solvent kinetic isotope effect determined for AAC(3)-IV (Figure S3), deviations of the Michaelis−Menten kinetics at high sisomycin concentrations (Figure S4), and ITC profiles of AAC(3)-IV with ribostamycin and with AcCoA (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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