Structural and Functional Characterization of the Recombinant Human Mitochondrial Trifunctional Protein
Structural and Functional Characterization of the Recombinant Human Mitochondrial Trifunctional Protein
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DOI：
10.1021/bi100742w
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发表时间：
2010-10-05
期刊：
影响因子：
2.9
通讯作者：
Arlaud, Gerard J.
中科院分区：
文献类型：
--
作者：
Fould, Benjamin;Garlatti, Virginie;Arlaud, Gerard J.
人线粒体三功能蛋白（TFP）的α和β亚基，参与脂肪酸β-氧化的多酶复合物，在大肠杆菌中共表达，并通过镍亲和层析纯化至均一。通过凝胶过滤、沉降速度和电子显微镜分析所得的α/His-β构建体，表明α（2）β（2）和α（4）β（4）复合物占优势，具有更高级的寡聚体。电子显微镜显示，基本物种α（2）β（2）与其细菌同源物具有总体结构相似性。如共沉降和表面等离子体共振分析所示，重组TFP与心磷脂和磷脂酰胆碱强烈相互作用，表明天然复合物通过与磷脂的直接相互作用与线粒体内膜相关联。重组TFP显示2-烯酰-CoA水合酶（ECH）、L-3-羟酰-CoA脱氢酶（HACD）和3-酮酰-CoA硫解酶（KACT）活性，并且当下游酶（分别为HACD和KACT）失活时，ECH和HACD各自达到平衡，表明反馈抑制。KACT活性在pH 9.5时最佳，对离子强度敏感，在底物3-酮十六烷酰-CoA浓度>5 μ M时受到抑制。其动力学常数（k（cat）= 169 s（-1）K-m = 4 μ M）与先前在纯化的猪TFP制备物上测定的一致。使用不同的测定，曲美他嗪，一种有效的抗阴道药，有没有显着的抑制作用的任何三个酶活性的重组TFP制剂，与其他报告相反。该研究首次提供了重组人TFP制剂的详细结构和功能表征，并为通过定点突变进行深入分析开辟了道路。
The alpha and beta subunits of the human mitochondrial trifunctional protein (TFP), the multienzyme complex involved in fatty acid beta-oxidation, were coexpressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography. The resulting alpha/His-beta construct was analyzed by gel filtration, sedimentation velocity, and electron microscopy, indicating a predominance of alpha(2)beta(2) and alpha(4)beta(4) complexes, with higher order oligomers. Electron microscopy indicated that the elementary species alpha(2)beta(2) had overall structural similarity with its bacterial homologue. As shown by cosedimentation and surface plasmon resonance analyses, recombinant TFP interacted strongly with cardiolipin and phosphatidylcholine, suggesting that the natural complex associates with the inner mitochondrial membrane through direct interactions with phospholipids. Recombinant TFP displayed 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD), and 3-ketoacyl-CoA thiolase (KACT) activities, and ECH and HACD each reached equilibrium when the downstream enzymes (HACD and KACT, respectively) were made inactive, indicating feed-back inhibition. The KACT activity was optimal at pH 9.5, sensitive to ionic strength, and inhibited at concentrations of its substrate 3-ketohexadecanoyl-CoA >5 mu M. Its kinetic constants (k(cat) = 169 s(-1) K-m = 4 mu M) were consistent with those determined previously on a purified porcine TFP preparation. Using different assays, trimetazidine, an efficient antiaginal agent, had no significant inhibitory effect on any of the three enzymatic activities of the recombinant TFP preparation, in contrast with other reports. This study provides the first detailed structural and functional characterization of a recombinant human TFP preparation and opens the way to in-depth analyses through site-directed mutagenesis.