Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase.
Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase.
复制标题
琥珀酸脱氢酶和琥珀酸泛醌还原酶的泛醌结合蛋白之间的相互作用。
DOI：
10.1016/0005-2728(80)90005-5
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
Yu,CA
中科院分区：
文献类型：
--
作者：
Yu,L;Yu,CA
琥珀酸-泛醌还原酶（QP）中纯化的泛醌结合蛋白与纯的可溶性琥珀酸脱氢酶在中性pH的磷酸盐缓冲液中混合后重构以形成琥珀酸-泛醌氧化还原酶。在琥珀酸脱氢酶与QP的重量比为约5时发现最大重构，这与计算值6.5相当接近，假定1摩尔QP与1摩尔琥珀酸脱氢酶反应而得到的值。当琥珀酸脱氢酶和QP被添加到复合物III或细胞色素b-c1 III复合物（高度纯化的泛喹啉-细胞色素还原酶）中时，琥珀酸-细胞色素还原酶被重建。重构的酶具有与天然酶复合物相同的动力学参数。QP和琥珀酸脱氢酶之间的相互作用导致后者的lowKm铁氰化物还原酶活性的消失。与可溶性琥珀酸脱氢酶不同，重组酶以及天然琥珀酸-细胞色素还原酶仅在过量泛醌存在下降低低浓度铁氰化物。泛醌对300 μM铁氰化物的表观Km值为6 μM，与泛醌作为电子受体时的Km值相近。当使用2，6_二氯酚靛酚作为电子受体用于重建琥珀酸酯-泛醌还原酶时，需要非常少或不需要外源泛醌来显示通过方法II制备的QP的最大活性，表明QP中结合的泛醌足以用于酶活性。除了恢复琥珀酸-泛醌还原酶活性之外，QP与琥珀酸脱氢酶之间的相互作用不仅稳定琥珀酸脱氢酶，而且部分解聚QP。重构的琥珀酸泛醌还原酶具有最小分子量为120000时，重构系统分散在0.2%的Triton X-100。在磷酸盐缓冲液、Tris-乙酸盐或Tris-磷酸盐缓冲液中，在中性pH下观察到最大重构。然而，Tris-HCl缓冲液产生的重构效率较低。这些结果表明，QP和琥珀酸脱氢酶之间的相互作用可能涉及一些阳离子基团，具有高亲和力的Cl-。QP的伯氨基不直接参与相互作用，因为当QP的氨基用荧光胺烷基化时，重构未显示出显著差异。重构的琥珀酸-泛醌还原酶的Arrhenius图显示，该酶在高于和低于26°C的温度下分别以19.7 kcal/mol和26.6 kcal/mol的活化能催化反应。这些活化能与天然酶获得的活化能相似。QP和琥珀酸脱氢酶之间相互作用的Arrhenius图也在26°C处具有断点。对于高于和低于折点的温度，这种相互作用的活化能计算为11.2千卡/摩尔和6.9千卡/摩尔。讨论中进一步探讨了酶促反应和重组反应活化能差异的意义。
Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochromecreductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochromeb-c1III complex (a highly purified ubiquinol-cytochromecreductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of lowKmferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochromecreductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparentKmfor ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to theKmwhen ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl−. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.