A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain α-ketoacid dehydrogenase complex
A synchronized substrate-gating mechanism revealed by cubic-core structure of the bovine branched-chain α-ketoacid dehydrogenase complex
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DOI：
10.1038/sj.emboj.7601444
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发表时间：
2006-12-13
期刊：
影响因子：
11.4
通讯作者：
Chuang, David T.
中科院分区：
文献类型：
--
作者：
Kato, Masato;Wynn, R. Max;Chuang, David T.
支链α-酮酸脱氢酶复合体的二氢硫胺酰基转移酶(E2b)组成立方体骨架，催化S-酰基二氢硫酰胺向辅酶A的酰基转移生成酰基-辅酶A。我们已经确定了哺乳动物(牛)E2b核心区的第一个晶体结构，该核心区有和没有结合的CoA或酰基CoA。这些结构揭示了组成立方核的两个相关三聚体之间的疏水和以前未报道的离子相互作用。在一个30埃长的活性中心通道中，二氢硫辛胺结合位点的入口在apo和acyl-CoA结合结构中是封闭的。CoA与通道的一个入口处的结合促进了通道的构象变化，导致相反的二氢硫胺门的打开。结合实验表明，CoA的存在显著提高了E2b核心与二氢硫胺的亲和力。这一结果支持了CoA结合是二氢硫胺门打开的原因的模型。我们认为这种门控机制使两种底物与活性中心通道的结合同步，活性中心通道作为前馈开关协调E2b催化的酰基转移反应。
The dihydrolipoamide acyltransferase (E2b) component of the branched-chain alpha-ketoacid dehydrogenase complex forms a cubic scaffold that catalyzes acyltransfer from S-acyldihydrolipoamide to CoA to produce acyl-CoA. We have determined the first crystal structures of a mammalian (bovine) E2b core domain with and without a bound CoA or acyl-CoA. These structures reveal both hydrophobic and the previously unreported ionic interactions between two-fold-related trimers that build up the cubic core. The entrance of the dihydrolipoamide-binding site in a 30-angstrom long active-site channel is closed in the apo and acyl-CoA-bound structures. CoA binding to one entrance of the channel promotes a conformational change in the channel, resulting in the opening of the opposite dihydrolipoamide gate. Binding experiments show that the affinity of the E2b core for dihydrolipoamide is markedly increased in the presence of CoA. The result buttresses the model that CoA binding is responsible for the opening of the dihydrolipoamide gate. We suggest that this gating mechanism synchronizes the binding of the two substrates to the active-site channel, which serves as a feed-forward switch to coordinate the E2b-catalyzed acyltransfer reaction.