Characterization of Molecular Defects in Isovaleryl-CoA Dehydrogenase in Patients with Isovaleric Acidemia

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• 作者：Al-Walid A. Mohsen ; Bambi D. Anderson ; Samuel L. Volchenboum ; Kevin P. Battaile ; Karen Tiffany ; David Roberts ; Jung-Ja P. Kim ; and Jerry Vockley
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：July 14, 1998
• 年：1998
• 卷：37
• 期：28
• 页码：10325 - 10335
• 全文大小：236K
• 年卷期：v.37,no.28(July 14, 1998)
• ISSN：1520-4995

文摘

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 K_m 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 170M^-1min^-1 for wild type.