Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity.
Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity.
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DOI：
10.1042/bj20040805
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发表时间：
2005-04
期刊：
影响因子：
--
通讯作者：
A. Tuganova;K. M. Popov
中科院分区：
文献类型：
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作者：
A. Tuganova;K. M. Popov
丙酮酸脱氢酶复合物（PDC）的转乙酰酶组分（E2）在调节丙酮酸脱氢酶激酶（PDHK）活性中起关键作用。本研究旨在进一步探讨E2调节PDHK活性的分子机制。与早期的结果一致，发现E2的内部L2（硫辛酰承载结构域2）与或不与C-末端铰链区一起表达对激酶活性几乎没有影响（如果有的话），表明L2对PDHK缺乏直接变构作用。与此形成鲜明对比的是，用由L2和E2的E1结合结构域（E1-binding domain）（L2-E1 BD双结构域）组成的构建体观察到PDHK的显著活化，这表明由E2结合促进的PDHK和E1的共定位和/或相互定向在很大程度上解释了转乙酰酶组分对PDHK的活化。等温滴定量热法和谷胱甘肽S-转移酶下拉试验表明，腺苷酸与PDHK分子的结合促进了L2结构域的释放。相反，L2结构域的结合导致PDHK对ATP的亲和力显著降低。腺苷酸和L2结构域与PDHK结合的串扰可能表明存在高度整合的机制，其中E2与ADP/ATP交换偶联的硫辛酰承载结构域与PDHK交换。
The transacetylase component (E2) of PDC (pyruvate dehydrogenase complex) plays a critical role in the regulation of PDHK (pyruvate dehydrogenase kinase) activity. The present study was undertaken to investigate further the molecular mechanism by which E2 modulates the activity of PDHK. In agreement with the earlier results, it was found that the inner L2 (lipoyl-bearing domain 2) of E2 expressed with or without the C-terminal hinge region had little, if any, effect on the kinase activity, indicating a lack of direct allosteric effect of L2 on PDHK. In marked contrast, significant activation of PDHK was observed with the construct consisting of L2 and the E1BD (E1-binding domain) of E2 (L2-E1BD didomain) suggesting that co-localization and/or mutual orientation of PDHK and E1, facilitated by E2 binding, largely account for the activation of PDHK by the transacetylase component. Isothermal titration calorimetry and glutathione S-transferase pull-down assays established that binding of adenyl nucleotides to the PDHK molecule facilitated the release of L2 domain. In contrast, binding of the L2 domain caused a significant decrease in the affinity of PDHK for ATP. The cross-talk in binding of adenyl nucleotides and the L2 domain to PDHK may indicate the existence of a highly integrated mechanism whereby the exchange of lipoyl-bearing domains presented to PDHK by E2 is coupled with ADP/ATP exchange.