Catalysis sensitive conformational changes in soybean lipoxygenase revealed by limited proteolysis and monoclonal antibody experiments.
Catalysis sensitive conformational changes in soybean lipoxygenase revealed by limited proteolysis and monoclonal antibody experiments.
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通过有限的蛋白水解和单克隆抗体实验揭示了大豆脂氧合酶的催化敏感构象变化。
DOI：
10.1021/bi00045a030
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
FunkJr,MO
中科院分区：
文献类型：
--
作者：
Ramachandran,S;Richards-Sucheck,TJ;Skrzypczak-Jankun,E;Wheelock,MJ;FunkJr,MO
大豆脂氧化酶催化多不饱和脂肪酸的脂质氢过氧化。通过有限的蛋白水解和一系列识别不连续表位并改变催化（抑制和激活）的单克隆抗体的组合，研究了配体介导的大豆脂氧合酶3 （L3）构象变化。胰蛋白酶将L3 （97 kDa）裂解为c端60 kDa和n端37 kDa片段。37 kDa的片段是从最初形成的38 kDa片段中获得的。利用蛋白足迹，抗体的表位被定位到37kda片段。在底物类似抑制剂油酸的存在下，蛋白水解仅产生60和38 kDa片段。即使经过长时间的孵育，38 kDa片段也没有进一步的蛋白水解。这不是一种洗涤剂效应，因为在SDS或Tween 20存在的情况下，蛋白质水解模式没有改变。在油酸存在的情况下，单克隆抗体与L3的结合显著减少，这为油酸-脂氧合酶相互作用诱导构象变化提供了额外的证据。这些观测是用最近解出的L3的三维结构来解释的。很明显，虽然该蛋白由一个小的n端/3桶结构域和一个主要为a-螺旋的大的c端结构域组成，但蛋白水解不会发生在两个结构域之间的连接区域。蛋白水解结果清楚地表明，较小的结构域在较大的结构域的整个长度上连接到一个狭窄的舌状突起，该突起延伸到提议的底物结合通道入口附近。有人提出，油酸结合时发生构象变化，这种变化通过蛋白质传递，改变了对蛋白质水解和抗体结合敏感的n端片段的结构特征。这是与脂氧合酶催化有关的构象变化的第一个直接证据。脂氧合酶在动植物多不饱和脂肪酸代谢中起着重要的作用。在许多哺乳动物细胞中，该酶负责从花生四烯酸开始生物合成白三烯、脂毒素和羟基二十碳四烯酸（Samuelsson等人，1987）。在植物中，氢过氧化的底物是亚油酸和亚麻酸，其产物是具有生长调节和抗虫害特性的化合物的生物合成的中间体（Siedow, 1991）。大豆脂氧化酶-1自1947年以来一直以高纯度形式存在（theorll et al., 1947）。
Soybean lipoxygenases catalyze lipid hydroperoxidation of polyunsaturated fatty acids. Putative ligand mediated conformational changes in soybean lipoxygenase 3 (L3) were studied by a combination of limited proteolysis and a series of monoclonal antibodies that recognize discontinuous epitopes and alter catalysis (inhibition and activation). Trypsin cleaved L3 (97 kDa) into C-terminal 60 kDa and N-terminal 37 kDa fragments. The 37 kDa fragment was obtained from a 38 kDa fragment formed initially. Using protein footprinting, the epitopes of the antibodies were mapped to the 37 kDa fragment. Proteolysis in the presence of a substrate analog inhibitor, oleic acid, generated the 60 and the 38 kDa fragments only. No further proteolysis of the 38 kDa fragment was seen even after prolonged incubation. This was not a detergent effect since the altered proteolysis pattern was not obtained in the presence of SDS or Tween 20. Binding of a monoclonal antibody to L3 in the presence of oleic acid was substantially reduced providing additional evidence for a conformational change induced by the oleic acid—lipoxygenase interaction. These observations are interpreted using the recently solved three-dimensional structure of L3. It is apparent that while the protein is composed of a small N-terminal/3-barrel domain and a large principally a-helical C-terminal domain, proteolysis does not take place at a linking region between the two domains. The proteolysis result makes it clear that the smaller domain is connected across the entire length of the larger domain to a narrow, tongue-like projection thatextends into the vicinity of the entrance to the proposed substrate binding channel. It is proposed that conformational changes take place upon oleic acid binding which are transmitted through the protein and alter structural features in the N-terminal fragment that are sensitive to proteolysis and antibody binding. This is the first direct evidence for conformational changes related to catalysis in lipoxygenase.Lipoxygenase plays a fundamental role in polyunsaturated fatty acid metabolism in plants and animals. The enzyme is responsible for the inauguration of the biosynthesis of leukotrienes, lipoxins, and hydroxyeicosatetraenoic acids from arachidonic acid in numerous mammalian cells (Samuelsson et al., 1987). In plants the substrates for hydroperoxidation are linoleic and linolenic acids, and the products are intermediates in the biosynthesis of compounds with growth regulatory as well as pest resistance properties (Siedow, 1991). Soybean lipoxygenase-1 has been available in a highly purified form since 1947 (Theorell et al., 1947),