Thermodynamic investigation of substrate activation and regulation in human short-chain acyl-CoA dehydrogenases

Human short-chain acyl-CoA dehydrogenase (hSCAD) is a flavoprotein that catalyzes the first step in the beta-oxidation cycle, producing up to 40% of the human energy requirement. Previous work with acyl-CoA dehydrogenases (ACDs) has shown these enzymes are specifically modulated upon binding of the substrate/product couple, allowing the reaction to proceed in a thermodynamically favorable manner. The act of binding the substrate/product couple has proven to be an essential factor for enzymatic function. The objective of this work is to examine the thermodynamic and biochemical properties of wild-type and mutant hSCAD enzymes to deconvolute the factors affecting the extent of substrate activation.; Wild-type hSCAD appears to be highly thermodynamically regulated in comparison to other ACDs studied. Specifically, the midpoint potential of the actual substrate/product couple shifts negative 33 mV when bound to the enzyme, allowing for electron transfer to occur from the acyl-CoA thioester to the flavin cofactor. This potential shift is over twice as large as that seen when the same ligand binds to the bacterial short-chain ACD and four times larger than that with mammalian medium-chain ACD. In order to further investigate this phenomenon an active site mutation, Glu368Gln, was constructed and its properties explored with natural thioester ligands. Studies with this kinetically dead mutant indicate that optimal substrate and product complexation generate similar enzyme potential shifts. Thus substrate has an equal, if not slightly greater, propensity towards thermodynamic modulation.; The consequences of inadequate electron transfer were probed using two naturally occurring disease-causing variants, Gly185Ser and Arg147Trp hSCAD. Arg147Trp appears to have similar redox properties to wild-type hSCAD, but mutations to the Gly 185 residue have dramatic effects on both the kinetic and thermodynamic properties. Although these mutations are not directly in the active site of hSCAD, Gly185Ser has impaired enzyme turnover and electron transfer is compromised by this mutation. The redox potential of substrate bound Gly185Ser actually shifts more negative and the potential of the substrate/product couple shifts more positive, thus making product formation more difficult. Studies with hSCAD will contribute to the overall knowledge of ACD systems as well as provide insight between bacterial and mammalian ACDs.