Thermal unfolding of medium-chain acyl-CoA dehydrogenase and iso(3)valeryl-CoA dehydrogenase: study of the effect of genetic defects on enzyme stability

AbstractGenetic defects affecting acyl-CoA dehydrogenases (ACAD)—key enzymes in the degradation of fatty acids and branched chain amino acids—are increasingly recognized as being more widespread than originally thought. For the medium-chain acyl-CoA dehydrogenase (MCAD), the K304E mutation is the most common genetic defect among Caucasian populations. The effect of substrate or substrate analog binding on the stability of wild-type MCAD and isovaleryl-CoA dehydrogenase (i3VD) and their genetic mutants (K304E- and T168A-MCAD and A282V-i3VD) is examined. Binding to the mutant ACADs is generally ≈10-fold weaker compared to wild-type proteins. Thermal stability of wt-MCAD (melting point ≈53.6 °C) is significantly higher compared to wt-i3VD (≈49.3 °C). With the exception of the A282V-i3VD mutant, a high degree of stabilization (5–11 °C) is induced by conversion into the reduced enzyme form complexed with product. The results are discussed based on the 3D-structures of the enzymes, and it is concluded that in the case of K304E-MCAD thermal stability as such is not a major contribution to the clinical phenotype. With the T168A-MCAD and A282V-i3VD mutants, however, the diminished thermal stability and minor stabilization by ligands must be regarded as an important factor contributing to the manifestation of the disease