The mitochondrial carnitine/acylcarnitine carrier (CACT) catalyzes an antiport of carnitine and acylcarnitines and also a uniport reaction with a rate of about one tenth with respect to the antiport rate. The antiport process results from the coupling of the two uniport reactions in opposite directions. In this mechanism, the transition of the carrier from the outward open conformation to the inward open one (or vice versa) is much faster for the carrier-substrate complex than for the unbound carrier. To investigate the molecular determinants that couple the binding of the substrate with the conformational transitions, site directed mutagenesis has been employed. The antiport or the uniport reaction was followed as [3H]carnitine uptake in or efflux from proteoliposomes reconstituted with the WT or Trp mutants of the rat CACT. Substitution of each the three Trp residues led to different results. Nearly no variations were observed upon substitution of W192 and/or W296 with Ala. While, substantial alteration of the transport function was observed in the mutants W224A, W224Y and W224F. Mutation of W224 led to the loss of the antiport function while the uniport function was unaltered. In these mutants impairment of the substrate affinity on the external side was also observed. The data highlights that W224 is involved in the coupling of the substrate binding with the matrix gate opening. The experimental data are in line with predictions by homology modeling of the CACT in its cytosolic (c-state) or matrix (m-state) opened conformations.

中文翻译：

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大鼠线粒体肉碱/酰基肉碱载体的色氨酸224对于反端口机制至关重要。

线粒体肉碱/酰基肉碱载体（CACT）催化肉碱和酰基肉碱的反转运，并且单向反应的速度约为反转运速率的十分之一。反端口过程是两个相反方向的单端口反应耦合产生的。在这种机制中，对于载体-底物复合物，载体从外向开放构象向内向开放构象的转变（反之亦然）要快于未结合载体。为了研究将底物的结合与构象转变偶联的分子决定簇，已经采用了定点诱变。进行反端口或单端口反应后，[3H]肉碱被大鼠CACT的WT或Trp突变体重组的脂质体吸收或流出。三个Trp残基中的每一个的取代导致不同的结果。W192和/或W296被Ala取代后几乎没有观察到变化，而在突变体W224A，W224Y和W224F中观察到转运功能的实质改变。W224的突变导致反端口功能的丧失，而单端口功能没有改变。在这些突变体中，还观察到了外侧上底物亲和力的损害。数据突出表明，W224参与了基板结合与矩阵门开口的耦合。实验数据与CACT的胞质（c状态）或基质（m状态）开放构象的同源性模型预测相符。在突变体W224A，W224Y和W224F中观察到转运功能的实质性改变。W224的突变导致反端口功能的丧失，而单端口功能没有改变。在这些突变体中，还观察到了外侧上底物亲和力的损害。数据突出表明，W224参与了基板结合与矩阵门开口的耦合。实验数据与CACT的胞质（c状态）或基质（m状态）开放构象的同源性模型预测相符。在突变体W224A，W224Y和W224F中观察到转运功能的实质性改变。W224的突变导致反端口功能的丧失，而单端口功能未改变。在这些突变体中，还观察到了外侧上底物亲和力的损害。数据突出表明，W224参与了基板结合与矩阵门开口的耦合。实验数据与CACT胞质（c状态）或基质（m状态）开放构象的同源性建模预测相符。数据突出表明，W224参与了基板结合与矩阵门开口的耦合。实验数据与CACT的胞质（c状态）或基质（m状态）开放构象的同源性模型预测相符。数据突出表明，W224参与了基板结合与矩阵门开口的耦合。实验数据与CACT的胞质（c状态）或基质（m状态）开放构象的同源性模型预测相符。