Thermodynamics of Ligand Binding and Catalysis in Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Comparative Studies Involving Normal and 3‘-Dephosphorylated C8-CoAs and Wild-Type and Asn191 → Ala (N191A) Mutant Enzymes†

Following our demonstration that the terminal 3‘-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751−760], we became interested in delineating its thermodynamic contribution in stabilizing the “ground” and “transition” state structures during enzyme catalysis. Since the 3‘-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3‘-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 → Ala (N191A) site-specific mutant enzymes. The experimental da...