Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex.
Characterization of interactions of dihydrolipoamide dehydrogenase with its binding protein in the human pyruvate dehydrogenase complex.
复制标题
人丙酮酸脱氢酶复合物中二氢硫辛酰胺脱氢酶与其结合蛋白相互作用的表征。
DOI：
10.1016/j.bbrc.2010.04.038
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发表时间：
2010
影响因子：
3.1
通讯作者：
Patel,MulchandS
中科院分区：
文献类型：
--
作者：
Park,Yun-Hee;Patel,MulchandS
与来自原核生物的丙酮酸脱氢酶复合物（PDC）不同，来自高等真核生物的PDC具有用于结合复合物中的二氢硫辛酰胺脱氢酶（E3）的额外结构组分E3结合蛋白（BP）。基于人（h）E3与hBP的双结构域（L3 S1）的亚复合物的3D结构，hE 3的用于与hBP结合的氨基酸残基（H348、D413、Y 438和R447）被丙氨酸或其它残基单独取代。当以其游离形式测量时，这些取代对hE 3活性没有大的影响。然而，当这些hE 3突变体在复合物中重构时，PDC活性显著降低，Y 438 A为9%，Y 438 H为20%，D413 A为18%。通过等温滴定量热法测定的hE 3突变体与L3 S1的结合显示，Y 438 A、Y 438 H和D413 A突变体与L3 S1的结合亲和力严重降低（分别为1019倍、607倍和402倍）。与野生型hE 3不同，Y 438 A突变体与L3 S1的结合伴随着不利的焓变和大的正熵变。这些结果表明hE 3-Y 438和hE 3-D413在hE 3与hBP的结合中起重要作用。
Unlike pyruvate dehydrogenase complexes (PDCs) from prokaryotes, PDCs from higher eukaryotes have an additional structural component, E3-binding protein (BP), for binding of dihydrolipoamide dehydrogenase (E3) in the complex. Based on the 3D structure of the subcomplex of human (h) E3 with the di-domain (L3S1) of hBP, the amino acid residues (H348, D413, Y438, and R447) of hE3 for binding to hBP were substituted singly by alanine or other residues. These substitutions did not have large effects on hE3 activity when measured in its free form. However, when these hE3 mutants were reconstituted in the complex, the PDC activity was significantly reduced to 9% for Y438A, 20% for Y438H, and 18% for D413A. The binding of hE3 mutants with L3S1 determined by isothermal titration calorimetry revealed that the binding affinities of the Y438A, Y438H, and D413A mutants to L3S1 were severely reduced (1019-, 607-, and 402-fold, respectively). Unlike wild-type hE3 the binding of the Y438A mutant to L3S1 was accompanied by an unfavorable enthalpy change and a large positive entropy change. These results indicate that hE3-Y438 and hE3-D413 play important roles in binding of hE3 to hBP.