Thermodynamics of aminoglycoside and acyl-coenzyme a binding to the Salmonella enterica AAC(6′)-Iy aminoglycoside N-acetyltransferase

Subray S. Hegde, Tarun K. Dam, C. Fred Brewer, John S. Blanchard

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39 Scopus citations


Kinetic and mechanistic studies on the chromosomally encoded aminoglycoside 6′-N-acetyltransferase, AAC(6′)-Iy, of Salmonella enterica that confers resistance toward aminoglycosides have been previously reported [Magnet et al. (2001) Biochemistry 40, 3700-3709]. In the present study, equilibrium binding and the thermodynamic parameters of binding of aminoglycosides and acyl-coenzyme A derivatives to AAC(6′)-Iy and of two mutants, C109A and the C109A/C70A double mutant, have been studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Association constants for different aminoglycosides varied greatly (4 × 104-150 × 104) while the association constants of several acyl-coenzyme A derivatives were similar (3.2 × 104-4.5 × 104). The association constants and van't Hoff enthalpy changes derived from intrinsic protein fluorescence changes were in agreement with independently measured values from isothermal titration calorimetry studies. Binding of both amino-glycosides and acyl-coenzyme A derivatives is strongly enthalpically driven and revealed opposing negative entropy changes, resulting in enthalpy-entropy compensation. The acetyltransferase exhibited a temperature-dependent binding of tobramycin with a negative heat capacity value of 410 cal mol-1 K-1. Isothermal titration studies of acetyl-coenzyme A and tobramycin binding to mutant forms of the enzyme indicated that completely conserved C109 does not play any direct role in the binding of either of the substrates, while C70 is directly involved in aminoglycoside binding. These results are discussed and compared with previous steady-state kinetic studies of the enzyme.

Original languageEnglish (US)
Pages (from-to)7519-7527
Number of pages9
Issue number23
StatePublished - Jun 11 2002

ASJC Scopus subject areas

  • Biochemistry


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