Isothermal titration microcalorimetric studies for the binding of octenoyl-CoA to medium chain acyl-CoA dehydrogenase.

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 degrees 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), delta G = -8.75 kcal/mol, delta H = -10.3 kcal/mol, and delta S = -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 delta G remains invariant as a function of temperature, delta H and deltaS(standard) both decrease with an increase in temperature. At temperatures of < 25 degrees C, delta G is dominated by favorable entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attain a value of zero at 23.8 degrees C, and then becomes unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of delta H yields the heat capacity change (delta Cp(0)) 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.