Kinetic mechanism of the GCN5-related chromosomal aminoglycoside acetyltransferase AAC(6')-Ii from Enterococcus faecium: evidence of dimer subunit cooperativity.
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.
Draker
Kari-ann
KA
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, 1200 Main Street West, Ontario L8N 3Z5, Canada.
Northrop
Dexter B
DB
Wright
Gerard D
GD
D000123:Acetyltransferases / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry; D002151:Calorimetry; D004058:Diffusion; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D016984:Enterococcus faecium / Q000201:enzymology* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D005810:Multigene Family; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000494:pharmacology; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D012997:Solvents; D014364:Tryptophan; D000123:Acetyltransferases; C056532:aminoglycoside N(6')-acetyltransferase; D000409:Alanine