Isothermal Titration Microcalorimetric Studies for the Binding of Octenoyl-CoA to Medium Chain Acyl-CoA Dehydrogenase

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Abstract
We investigated the binding of octenoyl-CoA to pig kidney medium chain acyl-CoA dehydrogenase (MCAD) by isothermal titration microcalorimetry under a variety of experimental conditions. At 25 °C in 50 mM phosphate buffer at pH 7.6 (ionic strength of 175 mM), the binding is characterized by the stoichiometry (n) of 0.89 mole of octenoyl-CoA/(mole of MCAD subunit), ΔG° = −8.75 kcal/mol, ΔH° = −10.3 kcal/mol, and ΔS° = −5.3 cal mol-1 K-1, suggesting that formation of MCAD−octenoyl-CoA is enthalpically driven. By employing buffers with various ionization enthalpies, we discerned that formation of the MCAD−octenoyl-CoA complex, at pH 7.6, accompanies abstraction (consumption) of 0.52 ± 0.15 proton/(MCAD subunit) from the buffer media. We studied the effects of pH, ionic strength, and temperature on the thermodynamics of MCAD−octenoyl-CoA interaction. Whereas the ionic strength does not significantly influence the above interaction, the pH of the buffer media exhibits a pronounced effect. The pH dependence of the association constant of MCAD + octenoyl-CoA ⇄ MCAD−octenoyl-CoA yields a pKa for the free enzyme of 6.2. Among thermodynamic parameters, whereas ΔG° remains invariant as a function of temperature, ΔH° and ΔS° both decrease with an increase in temperature. At temperatures of G° is dominated by favorable entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attain a value of zero at 23.8 °C, and then becomes unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy−entropy compensation. The temperature dependence of ΔH° yields the heat capacity change (ΔCp0) of −0.37 ± 0.05 kcal mol-1 K-1, attesting to the fact that the binding of octenoyl-CoA to MCAD is primarily dominated by the hydrophobic forces. The thermodynamic data presented herein are rationalized in light of structural−functional relationships in MCAD catalysis.