Biochemical and electrochemical characterization of two variant human short-chain acyl-CoA dehydrogenases.
Short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle with optimal activity toward butyryl- and hexanoyl-CoA. Two common variants of this enzyme encoding G185S and R147W substitutions have been identified at an increased frequency compared to the general population in patients with a wide variety of clinical problems, but functional studies of the purified mutant enzymes have shown only modestly changed kinetic properties. Moreover, both amino acid residues are located quite far from the catalytic pocket and the essential FAD cofactor. To clarify the potential relationship of these variants to clinical disease, we have further investigated their thermodynamic properties using spectroscopic and electrochemical techniques. Purified R147W hSCAD exhibited almost identical physical and redox properties to wild-type but only half of the specific activity and substrate activation shifts observed in wild-type enzyme. In contrast, the G185S mutant proved to have impairments of both its kinetic and electron transfer properties. Spectroelectrochemical studies reveal that G185S binding to the substrate/product couple produces an enzyme potential shift of only +88 mV, which is not enough to make the reaction thermodynamically favorable. For wild-type hSCAD, this barrier is overcome by a negative shift in the substrate/product couple midpoint potential, but in G185S this activation was not observed. When G185S was substrate bound, the midpoint potential of the enzyme actually shifted more negative. These results provide valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrate that mutations, regardless of their position in the protein structure, can have a large impact on the redox properties of the enzyme.
Saenger
Amy K
AK
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Kolthoff and Smith Halls, Minneapolis, Minnesota 55455, USA.
Nguyen
Tien V
TV
Vockley
Jerry
J
Stankovich
Marian T
MT
D044926:Butyryl-CoA Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D044926:Butyryl-CoA Dehydrogenase