Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric mitochondrial flavoenzyme whichcatalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. PCR of IVD genomic andcomplementary DNA was used to identify mutations occurring in patients with deficiencies in IVD activity.Western blotting, in vitro mitochondrial import, prokaryotic expression, and kinetic studies of IVD mutantswere conducted to characterize the molecular defects caused by the amino acid replacements. Mutationsleading to Arg21Pro, Asp40Asn, Ala282Val, Cys328Arg, Val342Ala, Arg363Cys, and Arg382Leureplacements were identified. Western blotting of fibroblast extracts and/or in vitro mitochondrial importexperiments indicate that the seven precursor IVD mutant peptides, and a previously identified IVDLeu13Pro mutant, are synthesized and imported into mitochondria. While the IVD Leu13Pro, Arg21Pro,and Cys328Arg mutant peptides are rapidly degraded following mitochondrial import, the other mutantpeptides exhibit greater mitochondrial stability, though less than the wild-type enzyme. Active IVDAla282Val, Val342Ala, Arg363Cys, and Arg382Leu mutants were less stable than wild type when producedin
Escherichia coli. The
Km values of purified IVD Ala282Val, Val342Ala, and Arg382Leu mutants are27.0, 2.8, and 6.9
M isovaleryl-CoA, respectively, compared to 3.1
M for the wild type, using theelectron-transfer flavoprotein (ETF) fluorescence quenching assay. The catalytic efficiency per mole ofFAD content of these three mutants is 4.8, 17.0, and 17.0
M
-1min
-1, respectively, compared to 170
M
-1min
-1 for wild type.