Energetics of two-step binding of a chromophoric reaction product, trans-3-indoleacryloyl-CoA, to medium-chain acyl-coenzyme-A dehydrogenase.

We previously demonstrated that the UV/visible spectrum of a chromophoric ligand, trans-3-indoleacryloyl-coenzyme-A (IACoA), is red-shifted (due to polarization of its carbonyl group) upon binding to pig kidney medium-chain acyl-CoA dehydrogenase (MCAD). The transient kinetic data revealed that the overall binding occurred in two steps. The first (fast) step involved the formation of an MCAD-IACoA collision complex in which the electronic structure of IACoA remained unchanged (the "colorless" complex), followed by a slow isomerization step with a concomitant red-shift in the IACoA spectrum (the "colored" complex) [Johnson, J. K., Wang, Z. X., and Srivastava, D. K. (1992) Biochemistry 31, 10564-10575]. To ascertain the energetics of the above two-step process, we investigated the temperature dependence of the spectral changes, binding constant of the MCAD-IACoA complex, and the rate constants for the conversion between the colorless and colored complexes. The data revealed that as the temperature of the incubation mixture of MCAD and IACoA ([IACoA] >> [MCAD] > Kd) increases from 12 to 35 degrees C, the resultant spectral peak of the MCAD-IACoA complex (lambda max = 417 nm) decreases. However, in this temperature range, the equilibrium constant for the second (isomerization) step remains unaffected. Isothermal titration calorimetric studies for the binding of IACoA to MCAD reveal that the overall binding energy at 25 degrees C (delta G degree = -7.4 kcal/mol) is contributed almost equally by the enthalpic (delta H degree = -3.7 kcal/mol) and entropic (T delta S degree = 3.7 kcal/mol) changes. As the temperature increases, both delta H degree and T delta S degree decrease proportionately, resulting in a strong enthalpy-entropy compensation effect. The temperature dependence of delta H degree yields a delta Cp degree value of -0.24 kcal/mol. The data presented herein throw light on the energetic consequences for the binding of IACoA to MCAD, the apparent similarity between the van't Hoff and calorimetric enthalpies, enthalpic and entropic contributions during the polarization of the carbonyl group of IACoA, and the overall structural-functional features of the enzyme-ligand complex as well as enzyme catalysis.