A high molecular weight dihydro-orotate dehydrogenase of Neurospora crassa. Purification and properties of the enzyme.
A high molecular weight dihydro-orotate dehydrogenase of Neurospora crassa. Purification and properties of the enzyme.
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粗糙脉孢菌的高分子量二氢乳清酸脱氢酶。
DOI：
10.1139/o75-175
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
R. Miller
中科院分区：
文献类型：
--
作者：
R. Miller
测定了粗糙脉孢菌（Neurospora crassa）中一种高分子量二氢乳清酸脱氢酶（DHOD）的一些不寻常的分子和催化性质。这种酶的性质与原核生物的可溶性生物合成酶的性质的比较揭示了几个重要的差异。真菌酶位于线粒体膜中与通过泛醌与呼吸链连接一致的位置（米勒，R.魏：生物化学，生物物理学。146，256-270（1971））。酶从膜上的释放导致含有结合的脂质和无活性疏水蛋白的溶解的蛋白质复合物。通过Triton-X-100和磷脂酶处理，在纯化过程中使非特异性蛋白质聚集最小化。催化活性酶的表观分子量为210 000。与可溶性DHOD制剂相反，高分子量酶没有内源性二氢乳清酸氧化酶（EC 1.3.3.1）活性，并且在二氢乳清酸（DHO）存在下对巯基反应性试剂的失活相对不敏感。该酶活性对引起黄素单甘肽（FMN）氧化的条件高度敏感。活性不能通过半胱氨酸或其他方式恢复。FMN在所有纯化制剂中均以结合、非荧光（还原）形式存在，直至二氢乳清酸被去除或氧化。纯化的酶的催化效率为12 000 mol DHO氧化每分钟每摩尔FMN。这种高周转率部分是由于纯化的酶的黄素含量小，相当于1摩尔FMN每120 000克的催化活性蛋白质。用原子吸收光谱法在纯化的酶中检测到铁，但不存在不稳定的硫化物。噻吩甲酰三氟丙酮，铁螯合剂，无论电子受体，只有部分抑制DHO氧化。脂肪酸与酶的疏水位点以非竞争性方式相互作用，但在某些条件下似乎显着改变泛醌的Km。相比之下，乳清酸是一种纯粹的竞争性抑制剂。这两种类型的抑制剂可以在体内调节乳清酸的生物合成。超氧阴离子不产生显着量的DHO还原酶，除非泛醌和一个合适的单电子载体，如吩嗪硫酸甲酯存在。已经提出DHOD是哺乳动物线粒体中超氧阴离子的来源（Forman，H. J. & Kennedy，J. A.：250，4322-4326（1975））。
Some of the unusual molecular and catalytic properties of a high molecular weight dihydro-orotate dehydrogenase (DHOD) from Neurospora crassa have been determined. Comparison of the properties of this enzyme with the properties of the soluble biosynthetic enzyme of prokaryotes has revealed several important differences. The fungal enzyme is located in a mitochondrial membrane in a position consistent with linkage with the respiratory chain through ubiquinone (Miller, R. W.: Arch. Biochem, Biophys. 146, 256-270 (1971)). Release of the enzyme from the membrane results in a solubilized protein complex containing bound lipids and inactive hydrophobic proteins. Non-specific protein aggregation is minimized during purification by Triton-X-100 and phospholipase treatments. The catalytically active enzyme has an apparent molecular weight of 210 000. In contrast to soluble DHOD preparations the high molecular weight enzyme has no endogenous dihydro-orotate oxidase (EC 1.3.3.1) activity and is relatively insensitive to inactivation by sulfhydryl-reactive reagents in the presence of dihydro-orotate (DHO). The enzyme activity is highly sensitive to conditions causing oxidation of flavin mononucleotide (FMN). The activity cannot be restored by cysteine or other means. FMN is present in all purified preparations in a bound, non-fluorescent (reduced) form until dihydro-orotic acid is removed or oxidized. Catalytic efficiency of the purified enzyme was 12 000 mol DHO oxidized per minute per mole FMN. This high turnover rate is due in part to the small flavin content of the purified enzyme, equivalent to 1 mol FMN per 120 000 g of catalytically active protein. Iron was detected in the purified enzyme by atomic absorption spectroscopy but labile sulfide was absent. Thenoyltrifluoroacetone, an iron chelator, only partially inhibited DHO oxidation regardless of electron acceptor. Fatty acids interact with a hydrophobic site of the enzyme in non-competitive fashion but under certain conditions appear to significantly alter the Km for ubiquinone. Orotate, by comparison, is a purely competitive inhibitor. Both types of inhibitor may function to regulate the biosynthesis of orotate in vivo. Superoxide anion is not produced in significant quantities by the DHO-reduced enzyme unless both ubiquinone and a suitable single electron carrier such as phenazine methosulfate are present. DHOD has been proposed as a source of superoxide anion in mammalian mitochondria (Forman, H. J. & Kennedy, J. A.: J. Biol. Chem. 250, 4322-4326 (1975)).