Localization of lipoyl-bearing domains in the alpha-ketoglutarate dehydrogenase multienzyme complex.
Localization of lipoyl-bearing domains in the alpha-ketoglutarate dehydrogenase multienzyme complex.
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α-酮戊二酸脱氢酶多酶复合物中硫辛酰基结构域的定位。
DOI：
10.1021/bi00310a001
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Frank,J
中科院分区：
文献类型：
--
作者：
Wagenknecht,T;Frank,J
Terence Wagenknecht* 和Joachim Frank摘要：来自大肠杆菌的α-酮戊二酸脱氢酶复合物由核心组分二氢硫辛酰转琥珀酰酶（E2）组成，其上非共价结合有α-酮戊二酸脱氢酶和二氢硫辛酰脱氢酶各自的12条多肽链。E2以包含24条相同链的立方体形状的复合物存在，并且可以从其他两种酶组分中解析。用胰蛋白酶对E2的有限消化定量地去除含有硫辛酸辅因子的结构域，同时保持复合物的四级结构完整。从负染色标本的电子显微镜照片获得的单分子图像计算天然和胰蛋白酶修饰的E2的Δ。这两个平均值非常相似，并且与先前通过X射线晶体学确定的模型基本一致。然而，通过从天然E2中减去胰蛋白酶处理的E2的平均值而获得的差异图像的详细分析显示，沿着天然分子的边缘沿着存在额外的染色排除区域，我们将其解释为代表硫辛酰承载域。同时分析了天然和修饰E2混合物的显微照片，以排除染色或电子光学伪影对结果的影响。基于这些结果沿着其他可用的结构信息，我们提出硫辛酰基结构域的一个功能是允许E2复合物中相距较远的硫辛酰基部分之间的相互作用;这一建议也与最近的生化数据建模研究结果一致[Hackert，M. L.，奥利弗河M.，& Reed，L. J.等人（1983）Proc. Acad. Sci. USA 80，2226-2230]，来自大肠杆菌的α-酮戊二酸脱氢酶多酶复合物（KGDC）1包含三种酶，它们都以多个拷贝存在（Pettit等人，1973）：12条α-酮戊二酸脱氢酶链（EC 1.2. 4.2）（E1），24条二氢硫辛酰转琥珀酰酶链（EC 2.3. 1.61）（E2）和12条二氢乙酰脱氢酶链（EC 1.6. 4.3）（E3）。这三种酶依次作用，催化以下总反应：
Terence Wagenknecht* and Joachim Frank abstract: The a-ketoglutarate dehydrogenase complex from Escherichia coli consists of a core component, dihydrolipoyl transsuccinylase (E2), to which are noncovalently bound 12 polypeptide chains each of a-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase. E2 exists as a cube-shaped complex comprising 24 identical chains and may be resolved from the other two enzyme components. Limited digestion of E2 with trypsin quantitatively removes domains containing the lipoic acid cofactor while leaving the quaternary structure of the complex intact. Averages of native and trypsin-modified E2 were computed from images of single molecules obtained from electron micrographs of negatively stained specimens. The two averages were very similar and were in general agreement with a model determined previously by X-ray crystallography. However, detailed analysis of the difference image, obtained by subtracting the average of the trypsintreated E2 from the native E2, showed extra stain-excluding regions along the edges of the native molecule which we interpret as representing the lipoyl-bearing domains. Micro-graphs of mixtures of native and modified E2 were also ana-lyzed in orderto rule out staining or electron-optical artifacts as accounting for the results. On thebasis of these results along with otheravailable structural information, we propose that one function of the lipoyl domains is to permit interactions between distantly separated lipoyl moieties in the E2 complex; this proposal alsoagrees with recent results of modeling studies of biochemical data [Hackert, M. L., Oliver, R. M., & Reed, L. J.(1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230], e a-ketoglutarate dehydrogenase multienzyme complex (KGDC) 1 from Escherichia coli comprises three enzymes which are all present in multiple copies (Pettit et al., 1973): 12 chains of a-ketoglutarate dehydrogenase (EC 1.2. 4.2)(El), 24 chains of dihydrolipoyl transsuccinylase (EC 2.3. 1.61)(E2), and 12 chains of dihydrolioyl dehydrogenase (EC 1.6. 4.3)(E3). The three enzymes, acting in sequence, catalyze the following overall reaction: