The kinetic mechanism of AAC(3)-IV aminoglycoside acetyltransferase from Escherichia coli

Maria L B Magalhaes, John S. Blanchard

Research output: Contribution to journalArticle

45 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)16275-16283
Number of pages9
JournalBiochemistry
Volume44
Issue number49
DOIs
StatePublished - Dec 13 2005

Fingerprint

Aminoglycosides
Escherichia coli
Kinetics
Kinetic parameters
Substrate Specificity
Substrates
Acetyltransferases
Isotopes
Catalysis
Anti-Bacterial Agents
Acids
aminoglycoside N(3')-acetyltransferase
aminoglycoside acetyltransferase
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

The kinetic mechanism of AAC(3)-IV aminoglycoside acetyltransferase from Escherichia coli. / Magalhaes, Maria L B; Blanchard, John S.

In: Biochemistry, Vol. 44, No. 49, 13.12.2005, p. 16275-16283.

Research output: Contribution to journalArticle

@article{cd3e6965e4dc4ffd89794f9005f4a398,
title = "The kinetic mechanism of AAC(3)-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.",
author = "Magalhaes, {Maria L B} and Blanchard, {John S.}",
year = "2005",
month = "12",
day = "13",
doi = "10.1021/bi051777d",
language = "English (US)",
volume = "44",
pages = "16275--16283",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "49",

}

TY - JOUR

T1 - The kinetic mechanism of AAC(3)-IV aminoglycoside acetyltransferase from Escherichia coli

AU - Magalhaes, Maria L B

AU - Blanchard, John S.

PY - 2005/12/13

Y1 - 2005/12/13

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=28944432885&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=28944432885&partnerID=8YFLogxK

U2 - 10.1021/bi051777d

DO - 10.1021/bi051777d

M3 - Article

C2 - 16331988

AN - SCOPUS:28944432885

VL - 44

SP - 16275

EP - 16283

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 49

ER -