The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate.
The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5'-phosphate.
复制标题
酵母线粒体柠檬酸转运蛋白：鉴定负责吡哆醛 5-磷酸介导的抑制作用的赖氨酸残基。
DOI：
10.1007/s10863-008-9187-1
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发表时间：
2008
影响因子：
3
通讯作者：
Kaplan,RonaldS
中科院分区：
文献类型：
--
作者：
Remani,Sreevidya;Sun,Jiakang;Kotaria,Rusudan;Mayor,JuneA;Brownlee,JuneM;Harrison,DavidHT;Walters,DEric;Kaplan,RonaldS
本研究确定了赖氨酸选择性试剂 5'-磷酸吡哆醛抑制线粒体内膜柠檬酸转运蛋白 (CTP) 功能的分子基础。动力学分析表明，PLP 是无 Cys CTP 的线性混合抑制剂，具有主要的竞争性成分。我们之前得出的结论是，CTP 至少包含两个底物结合位点，这些位点位于底物易位途径内越来越深的位置，并且包含关键的赖氨酸残基。在本研究中，通过七个赖氨酸到半胱氨酸取代突变体的功能表征，确定了底物结合位点一中的 Lys-83、底物结合位点二中的 Lys-37 和 Lys-239 以及其他四种旁路赖氨酸在赋予 PLP 运输抑制中的作用。我们观察到，用半胱氨酸替换 Lys-83 导致 PLP 介导的 CTP 功能抑制损失 78%。相比之下，用半胱氨酸替换 Lys-37 或 Lys-239 会导致 PLP 引起的抑制适度降低（即抑制作用分别损失 31% 和 20%）。有趣的是，这些 PLP 介导的抑制作用的损失可以通过用 MTSEA 对每个半胱氨酸进行共价修饰来挽救，MTSEA 是一种向半胱氨酸巯基添加赖氨酸样部分（即 SCH2CH2NH3+）的试剂。重要的是，用半胱氨酸替换非结合位点赖氨酸（即 Lys-45、Lys-48、Lys-134、Lys-141）导致 PLP 抑制几乎没有变化。基于这些结果，我们与CTP结构模型进行了对接计算，从而开发了PLP的物理结合模型。综合起来，我们的数据支持以下结论：PLP 通过与位于 CTP 的两个底物结合位点内的残基结合来发挥其主要抑制作用，其中 Lys-83 是总 PLP 效果的主要决定因素，因为替换该单个赖氨酸消除了几乎所有观察到的 PLP 抑制作用。
The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5′-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 insubstrate binding site one, Lys-37 and Lys-239 insubstrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH2CH2NH3+) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.