Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia
Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia
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DOI：
10.1021/bi973096r
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发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Vockley, J
中科院分区：
文献类型：
--
作者：
Mohsen, AA;Anderson, BD;Vockley, J
异戊酰辅酶A脱氢酶（IVD）是一种同源四聚体线粒体黄素酶，其催化异戊酰辅酶A转化为3-甲基巴豆酰辅酶A。IVD基因组和互补DNA的PCR用于鉴定IVD活性缺陷患者中发生的突变。蛋白质印迹，体外线粒体输入，原核表达，和动力学研究的IVD突变体进行了表征的氨基酸替换所造成的分子缺陷。鉴定了导致Arg 21 Pro、Asp 40 Asn、Ala 282 Val、Cys 328 Arg、Val 342 Ala、Arg 363 Cys和Arg 382 Leu置换的突变。成纤维细胞提取物和/或体外线粒体输入实验的蛋白质印迹法表明，七个前体IVD突变肽，和先前确定的IVD Leu 13 Pro突变体，合成和输入到线粒体。虽然IVD Leu 13 Pro，Arg 21 Pro和Cys 328 Arg突变肽在线粒体输入后迅速降解，但其他突变肽表现出更大的线粒体稳定性，尽管低于野生型酶。活性IVD Ala 282 Val、Val 342 Ala、Arg 363 Cys和Arg 382 Leu突变体在大肠杆菌中产生时不如野生型稳定。使用电子转移黄蛋白（ETF）荧光猝灭测定，纯化的IVD Ala 282 Val、Val 342 Ala和Arg 382 Leu突变体的K-m值分别为27.0、2.8和6.9 μ M异戊酰辅酶A，而野生型为3.1 μ M。这三种突变体的每摩尔FAD含量的催化效率分别为4.8、17.0和17.0 μ M-1.min（-1），而野生型为170 μ M-1.min（-1）。
Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric mitochondrial flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. PCR of IVD genomic and complementary 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 mutants were conducted to characterize the molecular defects caused by the amino acid replacements. Mutations leading to Arg21Pro, Asp40Asn, Ala282Val, Cys328Arg, Val342Ala, Arg363Cys, and Arg382Leu replacements were identified. Western blotting of fibroblast extracts and/or in vitro mitochondrial import experiments indicate that the seven precursor IVD mutant peptides, and a previously identified IVD Leu13Pro mutant, are synthesized and imported into mitochondria. While the IVD Leu13Pro, Arg21Pro, and Cys328Arg mutant peptides 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 in Escherichia coli. The K-m values of purified IVD Ala282Val, Val342Ala, and Arg382Leu mutants are 27.0, 2.8, and 6.9 mu M isovaleryl-CoA, respectively, compared to 3.1 mu M for the wild type, using the electron-transfer flavoprotein (ETF) fluorescence quenching assay. The catalytic 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.min(-1) for wild type.