Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis
Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis
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DOI：
10.1111/j.1399-3054.2004.00415.x
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发表时间：
2004-11-01
影响因子：
6.4
通讯作者：
Åkerlund, HE
中科院分区：
文献类型：
--
作者：
Gisselsson, A;Szilágyi, A;Åkerlund, HE
紫黄质脱环氧化酶（VDE）的调节涉及在低内腔pH下的构象变化，随后酶与类囊体膜结合。组氨酸残基在这个过程中的作用进行了研究，通过释放未结合的酶从类囊体超声处理后，在pH值范围为4.7至7.1。菠菜VDE（四个组氨酸）的膜结合的协同性被认为是3.8，相对于质子，并在pH 6.6的pH值下，而从小麦的VDE（三个组氨酸）显示出的协同性为2.9，并在pH 6.2的pH值下，有一个双离子点。构建了VDE的突变形式，并探测了它们与类囊体膜外部的结合。用一个或两个组氨酸取代丙氨酸或精氨酸后，与野生型相比，其协同性降低（1.6-2.3）。基于这些研究结果，组氨酸的pKa值是在VDE结合发生的范围内，我们建议，在低pH值的组氨酸残基的质子化诱导VDE的构象变化，从而间接调节酶的类囊体膜的结合。
Regulation of violaxanthin de-epoxidase (VDE) involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. The role of histidine residues in this process was studied by release of unbound enzyme from thylakoids upon sonication, on a pH scale from 4.7 to 7.1. The co-operativity for binding of spinach VDE (four histidines) to the membrane was found to be 3.8, with respect to protons, and had an inflexion point at pH 6.6, whereas VDE from wheat (three histidines) showed a co-operativity of 2.9 and had an inflexion point at pH 6.2. Mutant forms of VDE were constructed and probed for their binding to the outside of thylakoid membranes. With one or two histidines substituted for alanine or arginine, a lower co-operativity (1.6-2.3) was found, compared with the wild type. Based on these findings, and that the pKa value for histidine is within the range where the VDE binding takes place, we propose that protonation of the histidine residues at low pH induces the conformational change of VDE, and hence indirectly regulates binding of the enzyme to the thylakoid membrane.