Yang Y S, Datta A, Hainfeld J F, Furuya F R, Wall J S, Frey P A
Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705.
Biochemistry. 1994 Aug 16;33(32):9428-37. doi: 10.1021/bi00198a008.
This paper describes the organization of lipoyl moieties within the pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihydrolipoyl dehydrogenase. The core of the complex is the cubic 24-subunit E2 component, which contains the lipoyl moieties bonded to lipoyl-bearing domains. E1 and E3 are associated along the edges (E1) and on the faces (E3) of the core. The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex. The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E2). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of the E2E3 subcomplex is about half that of the PDH complex. The PDH complex and GC-PDH are both about 420 A in diameter, as determined by radial mass analysis, and the E2E3 subcomplex and GC-E2E3 are 320 and 350 A, respectively. The outer boundary of the E2E3 subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E2E3. Data obtained from radial mass analysis of GC-E2E3 and the unlabeled E2E3 subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E2 core. The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that many of the lipoyl groups in E2E3 subcomplexes relax outward into space vacated by the removal of E1 during the preparation of the subcomplex from PDH complex. Radial mass analysis of the PDH complex and GC-PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 A from the center of the complex, with the highest density at about 75 A from the particle centers, near the interface between E2 and the associated components E1 and E3.
本文描述了在扫描透射电子显微镜（STEM）中研究的大肠杆菌丙酮酸脱氢酶（PDH）复合物中硫辛酰部分的组织情况。PDH复合物是一种多酶复合物，由E1（丙酮酸脱氢酶）、E2（二氢硫辛酰转乙酰基酶）和E3（二氢硫辛酰脱氢酶）组成。该复合物的核心是由24个亚基组成的立方体型E2组分，其包含与含硫辛酰结构域相连的硫辛酰部分。E1和E3分别沿着核心的边缘（E1）和表面（E3）与之相连。硫辛酰部分用NADH还原，并用对马来酰亚胺基苯甲酰十一聚金簇复合物进行烷基化。发现金标记几乎特异性地与二氢硫辛酰转乙酰基酶（E2）结合。十一聚金簇通过STEM直接成像，并通过径向质量分析进行数字映射。E2E3亚复合物的质量约为PDH复合物的一半。通过径向质量分析确定，PDH复合物和GC - PDH的直径均约为420 Å，而E2E3亚复合物和GC - E2E3的直径分别为320 Å和350 Å。在GC - E2E3中，十一聚金标记清晰地显示了E2E3亚复合物的外边界。从GC - E2E3和未标记的E2E3亚复合物的径向质量分析获得的数据还表明，亚复合物的大小因围绕中央E2核心的含硫辛酰结构域而扩大。在从PDH复合物制备亚复合物的过程中，观察到E2E3亚复合物中的许多硫辛酰基团向外松弛到因去除E1而空出的空间中，这证实了硫辛酰部分能够通过含硫辛酰结构域中的结构移动进行长距离转运。对PDH复合物和GC - PDH的径向质量分析表明，硫辛酰基团分布在PDH复合物的很大区域，从中央核心延伸至复合物中心向外170 - 180 Å处，在距离颗粒中心约75 Å处密度最高，靠近E2与相关组分E1和E3的界面。
