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

Genetic 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 approximate to 10-fold weaker compared to wild-type proteins. Thermal stability of wt-MCAD (melting point approximate to 53.6degreesC) is significantly higher compared to wt-i3VD ( approximate to 49.3degreesC). With the exception of the A282V-i3VD mutant, a high degree of stabilization (5-11degreesC) 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. (C) 2004 Elsevier B.V. All rights reserved.