Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.
Loss of Complex I activity in the Escherichia coli enzyme results from truncating the C-terminus of subunit K, but not from cross-linking it to subunits N or L.
复制标题
大肠杆菌酶中复合物 I 活性的丧失是由于截短了 K 亚基的 C 末端，而不是由于将其与 N 或 L 亚基交联所致。
DOI：
10.1007/s10863-016-9655-y
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发表时间：
2016
影响因子：
3
通讯作者：
Vik,StevenB
中科院分区：
文献类型：
--
作者：
Zhu,Shaotong;Canales,Alejandra;Bedair,Mai;Vik,StevenB
复合体I是呼吸链的多亚单位酶，其膜臂有7个核心亚单位(A、H、J、K、L、M和N)。在大肠杆菌的酶中，K亚基的C端有10个氨基酸位于L亚基的侧螺旋上，并在细胞质表面形成K、L和N亚基的连接处。采用双半胱氨酸诱变方法，将K亚基(R99C)与L亚基(K581C)或N亚基(T292C)进行交联。部分交联物的产生对酶的活性或质子转运没有影响，表明K亚基的C末端在功能上没有动态作用。为了进一步阐明K亚基的作用，在C末端构建了基因缺失。通过对K亚基C末端最后4个残基的连续缺失，得到了不同的结果。缺失1个氨基酸对复合体I的活力影响不大，而缺失2个或2个以上氨基酸则导致膜囊泡制备过程中酶活性的完全丧失和L、M、N亚基水平的降低。综上所述，这些结果表明，虽然K亚基的C末端在复合体I的能量传递中没有动态作用，但它对酶的正确组装是至关重要的。
Complex I is a multi-subunit enzyme of the respiratory chain with seven core subunits in its membrane arm (A, H, J, K, L, M, and N). In the enzyme fromEscherichia colithe C-terminal ten amino acids of subunit K lie along the lateral helix of subunit L, and contribute to a junction of subunits K, L and N on the cytoplasmic surface. Using double cysteine mutagenesis, the cross-linking of subunit K (R99C) to either subunit L (K581C) or subunit N (T292C) was attempted. A partial yield of cross-linked product had no effect on the activity of the enzyme, or on proton translocation, suggesting that the C-terminus of subunit K has no dynamic role in function. To further elucidate the role of subunit K genetic deletions were constructed at the C-terminus. Upon the serial deletion of the last 4 residues of the C-terminus of subunit K, various results were obtained. Deletion of one amino acid had little effect on the activity of Complex I, but deletions of 2 or more amino acids led to total loss of enzyme activity and diminished levels of subunits L, M, and N in preparations of membrane vesicles. Together these results suggest that while the C-terminus of subunit K has no dynamic role in energy transduction by Complex I, it is vital for the correct assembly of the enzyme.