Specific Methylation of Asp160 (49 kDa subunit) Located inside the Quinone Binding Cavity of Bovine Mitochondrial Complex I
Specific Methylation of Asp160 (49 kDa subunit) Located inside the Quinone Binding Cavity of Bovine Mitochondrial Complex I
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DOI：
10.1021/acs.biochem.6b00190
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发表时间：
2016-06-14
期刊：
影响因子：
2.9
通讯作者：
Miyoshi, Hideto
中科院分区：
文献类型：
--
作者：
Murai, Masatoshi;Inaoka, Hiroyuki;Miyoshi, Hideto
Asp160位于牛线粒体复合体I的49 kDa亚基上，位于醌结合腔的内部，被认为是酶能量转化的重要残基。为了阐明该残基的催化功能，我们试图通过配体定向tosyl （LDT）化学技术将49 kDa Asp160 [Asp(COO)-CH3]以乙酰素衍生物（ALM）作为高亲和力甲基化。我们通过液相色谱-质谱分析49 kDa亚基的胰蛋白酶消化证实了49 kDa Asp160的特异性甲基化。占据醌结合腔的喹唑啉类抑制剂（[I-125]AzQ）的结合亲和力不受甲基化的影响，表明这种化学修饰不会引起醌结合腔内的显著结构变化。49 kDa的Asp160甲基化未导致催化活性完全丧失；修饰后的酶保留了部分电子转移和质子易位活性。这些结果以及49 kDa Asp160在局部蛋白质环境中对各种LDT试剂具有很强的亲核性这一事实强烈表明，该残基不受附近残基产生的严格相互作用（如静电相互作用）的影响，在功能上很重要，但对复合物I的能量转换不是必需的。
Asp160 in the 49 kDa subunit of bovine mitochondrial complex I, which is located in the inner part of the quinone binding cavity, is considered to be an essential residue for energy conversion of the enzyme. To elucidate the catalytic function of this residue, we attempted to specifically methylate 49 kDa Asp160 [Asp(COO)-CH3] through a ligand-directed tosyl (LDT) chemistry technique with an acetogenin-derivative (ALM) as A high-affinity We confirmed the specific methylation of 49 kDa Asp160 through liquid chromatography tandem mass spectrometry analysis of the tryptic digests of the 49 kDa subunit. The binding affinity of a quinazoline-type inhibitor ([I-125]AzQ) occupying the quinone binding cavity was not affected by methylation, indicating that this chemical modification does not induce significant structural changes inside the quinone binding cavity. The methylation of 49 kDa Asp160 did not lead to the complete loss of catalytic activity; the modified enzyme retained partial electron transfer and proton translocation activities. These results along with the-fact that 49 kDa Asp160 elicits a very strong nucleophilicity against,various LDT reagents in the local protein environment strongly suggest that this residue is free from strict interactions (such as electrostatic interaction) arising from nearby residue(s) and is functionally important but not essential for the energy conversion of complex I.