Packman L C, Perham R N
Biochem J. 1987 Mar 1;242(2):531-8. doi: 10.1042/bj2420531.
The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented.
通过有限蛋白酶解研究了大肠杆菌2-氧代酸脱氢酶复合物中二氢硫辛酰胺酰基转移酶(E2)组分的结构。用胰蛋白酶和金黄色葡萄球菌V8蛋白酶从丙酮酸脱氢酶复合物的E2p组分中切除三个硫辛酰结构域,并从2-氧代戊二酸脱氢酶复合物的E2o组分中切除单个硫辛酰结构域。通过对分离的硫辛酰片段以及截短的E2p和E2o链进行序列分析,确定了这些酶在每个E2链上的主要作用位点。众多切割位点中的每一个(E2p中有12个,E2o中有6个)都位于E2链的相似片段内,即富含丙氨酸、脯氨酸和/或带电荷氨基酸的多肽片段。这些区域显然可被分子量为24,000 - 28,000的蛋白酶接近,并且根据核磁共振光谱,其中一些区域先前因其构象灵活性而与促进结构域运动有关。有限蛋白酶解数据表明,E2p和E2o具有比通过检查其氨基酸序列所预测的更紧密的结构相似性。这项工作的结果是,在根据先前发表的编码基因sucB的序列推断的E2o序列中检测到一个错误。通过直接方法对来自E2o的相关肽进行了纯化和测序;给出了修正后的序列。