CHARACTERIZATION OF MOLECULAR DEFECTS IN ISOVALERYL-COA DEHYDROGENASEIN PATIENTS WITH ISOVALERIC ACIDEMIA

Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric mitochondrial f lavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methy lcrotonyl-CoA. PCR of IVD genomic and complementary DNA was used to id entify mutations occurring in patients with deficiencies in IVD activi ty. Western blotting, in vitro mitochondrial import, prokaryotic expre ssion, and kinetic studies of IVD mutants were conducted to characteri ze the molecular defects caused by the amino acid replacements. Mutati ons leading to Arg21Pro, Asp40Asn, Ala282Val, Cys328Arg, Val342Ala, Ar g363Cys, and Arg382Leu replacements were identified. Western blotting of fibroblast extracts and/or in vitro mitochondrial import experiment s indicate that the seven precursor IVD mutant peptides, and a previou sly identified IVD Leu13Pro mutant, are synthesized and imported into mitochondria. While the IVD Leu13Pro, Arg21Pro, and Cys328Arg mutant p eptides are rapidly degraded following mitochondrial import, the other mutant peptides exhibit greater mitochondrial stability, though less than the wild-type enzyme. Active IVD Ala282Val, Val342Ala, Arg363Cys, and Arg382Leu mutants were less stable than wild type when produced i n Escherichia coli. The K-m values of purified IVD Ala282Val, Val342Al a, and Arg382Leu mutants are 27.0, 2.8, and 6.9 mu M isovaleryl-CoA, r espectively, compared to 3.1 mu M for the wild type, using the electro n-transfer flavoprotein (ETF) fluorescence quenching assay. The cataly tic efficiency per mole of FAD content of these three mutants is 4.8, 17.0, and 17.0 mu M-1.min(-1), respectively, compared to 170 mu M-1.mi n(-1) for wild type.