Komuniecki R, Fekete S, Thissen-Parra J
J Biol Chem. 1985 Apr 25;260(8):4770-7.
The 2-methyl branched-chain acyl-CoA dehydrogenase was purified to homogeneity from mitochondria of the parasitic nematode, Ascaris suum. The native molecular weight of the enzyme was estimated to be 170,000 by gel filtration. The enzyme migrated as a single protein band with Mr = 42,500 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the enzyme is a tetramer composed of identical subunits. The enzyme exhibited absorbance maxima at 272, 375, and 452 with a ratio 7.9:0.8:1.0, respectively. FAD content was estimated to be 0.9 mol/mol of subunit and the absorption coefficient of FAD at 452 nm was 14.1 mM-1 cm-1. The purified enzyme dehydrogenated both 2-methylbutyryl-CoA and 2-methylvaleryl-CoA with apparent Km and Vmax values of 18 microM and 1.62 mumol/min/mg and 21 microM and 1.58 mumol/min/mg, respectively. This enzyme also appeared to dehydrogenate butyryl-CoA, valeryl-CoA, and octanoyl-CoA but at a much lower rate. The enzyme did not dehydrogenate propionyl-CoA, isobutyryl-CoA, isovaleryl-CoA, and palmitoyl-CoA. Tiglyl-CoA and 2-methyl-2-pentenoyl-CoA were identified as reaction products from 2-methylbutyryl- and 2-methylvaleryl-CoA, respectively. Dehydrogenating activity with both substrates was inhibited by tiglyl-CoA, acetoacetyl-CoA, and straight chain acyl CoAs of increasing chain length. N-Ethylmaleimide and p-hydroxymercuribenzoate had little effect on dehydrogenating activity but the heavy metals Hg2+ and Ag2+ were potent inhibitors. Physiologically, the dehydrogenase functions as a branched-chain enoyl-CoA reductase. Incubations of A. suum submitochondrial particles, NADH, tiglyl-CoA, purified A. suum electron-transfer flavoprotein, and the 2-methyl branched-chain acyl-CoA dehydrogenase resulted in the rotenone-sensitive, dehydrogenase-dependent formation of 2-methylbutyryl-CoA.
从寄生线虫猪蛔虫的线粒体中纯化出了具有同质性的2-甲基支链酰基辅酶A脱氢酶。通过凝胶过滤法估计该酶的天然分子量为170,000。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳过程中，该酶以单一蛋白条带迁移，Mr = 42,500，这表明该酶是由相同亚基组成的四聚体。该酶在272、375和452处呈现吸光度最大值，其比例分别为7.9:0.8:1.0。FAD含量估计为每摩尔亚基0.9摩尔，FAD在452nm处的吸收系数为14.1 mM-1 cm-1。纯化后的酶使2-甲基丁酰辅酶A和2-甲基戊酰辅酶A脱氢，其表观Km和Vmax值分别为18 microM和1.62 mumol/min/mg以及21 microM和1.58 mumol/min/mg。该酶似乎也能使丁酰辅酶A、戊酰辅酶A和辛酰辅酶A脱氢，但速率要低得多。该酶不能使丙酰辅酶A、异丁酰辅酶A、异戊酰辅酶A和棕榈酰辅酶A脱氢。分别鉴定出巴豆酰辅酶A和2-甲基-2-戊烯酰辅酶A是2-甲基丁酰辅酶A和2-甲基戊酰辅酶A的反应产物。两种底物的脱氢活性均受到巴豆酰辅酶A、乙酰乙酰辅酶A以及链长增加的直链酰基辅酶A的抑制。N-乙基马来酰亚胺和对羟基汞苯甲酸对脱氢活性影响不大，但重金属Hg2+和Ag2+是强效抑制剂。在生理上，该脱氢酶起支链烯酰辅酶A还原酶的作用。猪蛔虫亚线粒体颗粒、NADH、巴豆酰辅酶A、纯化的猪蛔虫电子传递黄素蛋白和2-甲基支链酰基辅酶A脱氢酶一起孵育，导致了鱼藤酮敏感的、依赖脱氢酶的2-甲基丁酰辅酶A的形成。
