Source:http://linkedlifedata.com/resource/pubmed/id/12767240
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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
21
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pubmed:dateCreated |
2003-5-27
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pubmed:abstractText |
The aminoglycoside 6'-N-acetyltransferase AAC(6')-Ii from Enterococcus faecium is an important microbial resistance determinant and a member of the GCN5-related N-acetyltransferase (GNAT) superfamily. We report here the further characterization of this enzyme in terms of the kinetic mechanism of acetyl transfer and identification of rate-contributing step(s) in catalysis, as well as investigations into the binding of both acetyl-CoA and aminoglycoside substrates to the AAC(6')-Ii dimer. Product and dead-end inhibition studies revealed that AAC(6')-Ii follows an ordered bi-bi ternary complex mechanism with acetyl-CoA binding first followed by antibiotic. Solvent viscosity studies demonstrated that aminoglycoside binding and product release govern the rate of acetyl transfer, as evidenced by changes in both the k(cat)/K(b) for aminoglycoside and k(cat), respectively, with increasing solvent viscosity. Solvent isotope effects were consistent with our viscosity studies that diffusion-controlled processes and not the chemical step were rate-limiting in drug modification. The patterns of partial and mixed inhibition observed during our mechanistic studies were followed up by investigating the possibility of subunit cooperativity in the AAC(6')-Ii dimer. Through the use of AAC-Trp(164) --> Ala, an active mutant which exists as a monomer in solution, the partial nature of the competitive inhibition observed in wild-type dead-end inhibition studies was alleviated. Isothermal titration calorimetry studies also indicated two nonequivalent antibiotic binding sites for the AAC(6')-Ii dimer but only one binding site for the Trp(164) --> Ala mutant. Taken together, these results demonstrate subunit cooperativity in the AAC(6')-Ii dimer, with possible relevance to other oligomeric members of the GNAT superfamily.
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pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Acetyltransferases,
http://linkedlifedata.com/resource/pubmed/chemical/Alanine,
http://linkedlifedata.com/resource/pubmed/chemical/Solvents,
http://linkedlifedata.com/resource/pubmed/chemical/Tryptophan,
http://linkedlifedata.com/resource/pubmed/chemical/aminoglycoside...
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pubmed:status |
MEDLINE
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pubmed:month |
Jun
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pubmed:issn |
0006-2960
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
3
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pubmed:volume |
42
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
6565-74
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pubmed:dateRevised |
2006-11-15
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pubmed:meshHeading |
pubmed-meshheading:12767240-Acetyltransferases,
pubmed-meshheading:12767240-Alanine,
pubmed-meshheading:12767240-Calorimetry,
pubmed-meshheading:12767240-Diffusion,
pubmed-meshheading:12767240-Dimerization,
pubmed-meshheading:12767240-Dose-Response Relationship, Drug,
pubmed-meshheading:12767240-Enterococcus faecium,
pubmed-meshheading:12767240-Escherichia coli,
pubmed-meshheading:12767240-Kinetics,
pubmed-meshheading:12767240-Models, Chemical,
pubmed-meshheading:12767240-Multigene Family,
pubmed-meshheading:12767240-Mutation,
pubmed-meshheading:12767240-Protein Binding,
pubmed-meshheading:12767240-Protein Structure, Tertiary,
pubmed-meshheading:12767240-Solvents,
pubmed-meshheading:12767240-Thermodynamics,
pubmed-meshheading:12767240-Tryptophan
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pubmed:year |
2003
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pubmed:articleTitle |
Kinetic mechanism of the GCN5-related chromosomal aminoglycoside acetyltransferase AAC(6')-Ii from Enterococcus faecium: evidence of dimer subunit cooperativity.
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pubmed:affiliation |
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, 1200 Main Street West, Ontario L8N 3Z5, Canada.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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