title: The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli.
abstract: 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Maria L B
authors.initials: ML
authors.lastname: Magalhaes
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002151:Calorimetry; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007554:Isotopes / Q000737:chemistry; D015394:Molecular Structure; D009328:Nebramycin / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D007554:Isotopes; D009328:Nebramycin; C011666:apramycin; D000123:Acetyltransferases; C045539:aminoglycoside N(3')-acetyltransferase