Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C.
Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C.
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发表时间：
1989-01
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通讯作者：
M. Rahmatullah;S. Gopalakrishnan;G. Radke;T. Roche
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作者：
M. Rahmatullah;S. Gopalakrishnan;G. Radke;T. Roche
二氢硫辛酰转乙酰基酶-蛋白X-激酶亚复合体(E_2-X-KcKb)用精氨酸酶C处理，选择性地将蛋白X转化为内区片段(MR约等于35,000)和外区(MR约等于15,500)。这些片段分别比来自E2组分的内区片段和外区片段大和小，支持了蛋白质X不同于E2组分的结论。蛋白酶Arg C切割KB亚基的速度比蛋白X慢。与KB亚基的这种切割相关的激酶活性的增加。更慢的E2亚基的裂解产生内域片段(MR约等于31,500)和含硫辛基的结构域片段(MR约等于49,000)，其MR值分别比通过胰酶处理亚复合体产生的相应E2片段大至少3,000和10,000。用精氨酸酶或胰酶进行不同程度的切割后，分离并鉴定了残留的寡聚亚复合体。我们发现，选择性地去除蛋白X的硫酰基结构域并不改变脂酰基对激酶的调节作用，这表明与E2亚基结合的硫酰残基是有效的，在完全去除E2和蛋白X的外部结构域后，蛋白X的内域仍然与E2亚基的内域相关，只有10%的E2亚基完整，近一半的催化(KC)亚基被残留亚复合体结合，而从E2亚基中去除剩余的外部结构域释放了KC亚基。因此，蛋白X在复合体的亚基中是独一无二的，它与转乙酰基酶的寡聚内部结构域紧密结合，而转乙酰基酶的外部结构域用于结合并促进对激酶催化亚基的调节。
Treatment of the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2-X-KcKb) with protease Arg C selectively converted protein X into an inner domain fragment (Mr approximately equal to 35,000) and an outer (lipoyl-bearing) domain fragment (Mr approximately equal to 15,500). These fragments were larger and much smaller, respectively, than the inner domain and outer domain fragments derived from the E2 component, supporting the conclusion that protein X is distinct from the E2 component. Protease Arg C cleaved the Kb subunit more slowly than protein X. An increase in kinase activity correlated with this cleavage of the Kb subunits. An even slower cleavage of E2 subunits generated an inner domain fragment (Mr approximately equal to 31,500) and a lipoyl-bearing domain fragment (Mr approximately equal to 49,000) which had Mr values at least 3,000 and 10,000 larger, respectively, than the corresponding E2 fragments generated by trypsin treatment of the subcomplex. Following various extents of cleavage with protease Arg C or trypsin, residual oligomeric subcomplexes were isolated and characterized. We found that selective removal of the lipoyl-bearing domain of protein X did not alter lipoyl-mediated regulation of the kinase indicating that the lipoyl residues bound to E2 subunits are effective, that the inner domain of protein X remained associated with the inner domain of E2 subunits following the complete removal of the outer domains of both E2 and protein X, that, with only 10% of the E2 subunits intact, nearly half of the catalytic (Kc) subunits of the kinase were bound by the residual subcomplex, and that removal of the remaining outer domains from E2 subunits released the Kc subunits. Thus, protein X is unique among the subunits of the complex in binding tightly to the oligomeric inner domain of the transacetylase, and the outer domain of the transacetylase serves to bind to and facilitate the regulation of the catalytic subunit of the kinase.