THERMODYNAMICS OF LIGAND-BINDING AND CATALYSIS IN HUMAN LIVER MEDIUM-CHAIN ACYL-COA DEHYDROGENASE - COMPARATIVE-STUDIES INVOLVING NORMAL AND 3'-DEPHOSPHORYLATED C-8-COAS AND WILD-TYPE AND ASN191-]ALA(N191A) MUTANT ENZYMES

Following our demonstration that the terminal 3'-phosphate group of ac yl-CoA substrates (which is confined to the exterior of the protein st ructure, and is fully exposed to the outside solvent environment) exhi bits a functional role in the recombinant human liver medium-chain acy l-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Sr ivastava, D. K. (1997) Biochem. J. 325, 751-760], we became interested in delineating its thermodynamic contribution in stabilizing the ''gr ound'' and ''transition'' state structures during enzyme catalysis. Si nce the 3'-phosphate group of the coenzyme A thiolester has the potent ial to form a hydrogen bond with the side chain group of Asn-191, thes e studies were performed utilizing both normal and 3'-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C -8-CoA) as the physiological substrate and product of the enzyme, resp ectively, as well as utilizing wild-type and Asn191 --> Ala (N191A) si te-specific mutant enzymes. The experimental data revealed that the en thalpic contribution of the 3'-phosphate group was similar in both gro und and transition states, and was primarily derived from the London-v an der Waals interactions (between the 3'-phosphate group of C-8-CoA a nd the surrounding protein moiety), rather than from the potential hyd rogen bonding. The temperature dependence of Delta H degrees for the b inding of octenoyl-CoA and 3'-dephosphooctenoyl-CoA revealed that the deletion of the 3'-phosphate group from octenoyl-CoA increased the mag nitude of the heat capacity changes (Delta C(p)degrees) from -0.53 to -0.59 kcal mol(-1) K-1. Although the latter effect could be attributed to an increase in the relative hydrophobicity of the Ligand, the expe rimentally observed Delta C(p)degrees's (for either of the Ligands) co uld not be predicted on the basis of the changes in the solvent-access ible surface areas of the enzyme and ligand species. These coupled wit h the fact that the Delta C(p)degrees for the binding of octenoyl-CoA to pig kidney MCAD (which is believed to be structurally identical to human liver MCAD) is only -0.37 kcal mol(-1) K-1 [Srivastava, D. K., W ang, S., and Peterson, K. L. (1997) Biochemistry 36, 6359-6366] prompt us to question the reliability of predicting the Delta C(p)degrees va lues of the enzyme-ligand complexes from their X-ray crystallographic data. Arguments are presented that certain intrinisic limitations of t he crystallographic data preclude kinetic and thermodynamic prediction s about the enzyme-ligand complexes and enzyme catalysis.