Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative
Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative
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DOI：
10.1074/jbc.m110.104547
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发表时间：
2010-08-20
影响因子：
4.8
通讯作者：
Imanaka, Tsuneo
中科院分区：
文献类型：
--
作者：
Kashiwayama, Yoshinori;Tomohiro, Takenori;Imanaka, Tsuneo
过氧化物酶体在包括脂肪酸及其衍生物的β-氧化在内的许多重要代谢途径中发挥重要作用。因此，过氧化物酶体具有各种β-氧化酶和专门的脂肪酸转运系统。然而，这些蛋白质的分子机制，特别是在底物结合方面，仍然是未知的。在这项研究中，以确定这些蛋白质的底物结合位点，我们合成了一个光反应性棕榈酸类似物轴承作为载体的diazirine部分，并进行光亲和标记纯化的大鼠肝脏过氧化物酶体。其结果是，一个80 kDa的过氧化物酶体蛋白特异性标记的光亲和配体，并在棕榈酰辅酶A的存在下，标记效率竞争性下降。质谱分析鉴定了80 kDa的蛋白为过氧化物酶体多功能酶2型（MFE 2），过氧化物酶体β-氧化酶之一。重组大鼠MFE 2也被光亲和配体标记，质谱分析显示大鼠MFE 2的片段（残基Trp（249）至Arg（251））被配体标记。带有这些残基的MFE 2突变体MFE 2（W249 A）和MFE 2（R251 A）表现出降低的标记效率。此外，对应于人MFE 2中的致病突变之一的MFE 2（W249 G）也表现出降低的效率。基于大鼠MFE 2的晶体结构，这些残基位于导致MFE 2活性位点的疏水腔的顶部。这些数据表明MFE 2将其底物锚定在从Trp（249）到Arg（251）的区域周围，并将底物沿着疏水空腔以朝向催化中心的适当方向定位。
Peroxisomes play an essential role in a number of important metabolic pathways including beta-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various beta-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal beta-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp(249) to Arg(251)) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp(249) to Arg(251) and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.