Mobility of polypeptide chain in the pyruvate dehydrogenase complex revealed by proton NMR
Mobility of polypeptide chain in the pyruvate dehydrogenase complex revealed by proton NMR
复制标题
质子核磁共振揭示丙酮酸脱氢酶复合物中多肽链的迁移率
作者：
R. Perham;H. Duckworth;G. Roberts
最近通过NMR光谱学和X射线晶体学对几种小蛋白质的研究已经清楚地证明了它们结构中显著的内部移动性（参见例如参考文献1-9），其不仅可以涉及氨基酸侧链，而且可以涉及多肽链的较大区域。偶尔，这种流动性的一个合理的功能已被证实1，9，但一直没有确凿的证据，分子内流动性和酶机制中的一个定义步骤之间的直接联系。大肠杆菌的丙酮酸脱氢酶（PDH）多酶复合物（分子量（Mr）4.5-6 ×106）是已知最大的明确定义的蛋白质组装体之一，包含三种不同酶的多个拷贝10，11。底物通过硫辛酸乙酰转移酶组分（复合物的结构核心）的硫辛酰-赖氨酸残基以硫酯键进行。硫辛酰-赖氨酸残基充当摆动臂，在三种酶的催化中心之间12 -15以及在与硫辛酸乙酰转移酶核心中的不同亚基连接的硫辛酸残基之间16 -18携带底物。已经证实，多肽链的含硫辛酸区域可能是柔性的19，20，因此能够大大增加摆动臂的有效半径19。我们在这里报告意想不到的尖锐线在270 MHz的质子NMR谱的酶复合物，这是由于显着的构象流动性大区域的多肽链携带的硫辛酸残基。这种移动性将增强多亚基结构中活性位点的功能连接。
Recent studies of several small proteins by NMR spectroscopy and X-ray crystallography have clearly demonstrated significant internal mobility in their structures (see, for example, refs 1–9), which can involve not only amino acid side chains but also larger regions of polypeptide chain. Occasionally a plausible function for this mobility has been suggested1,9, but there has been no conclusive evidence for a direct connection between intramolecular mobility and a defined step in an enzymatic mechanism. The pyruvate dehydrogenase (PDH) multienzyme complex of Escherichia coli (molecular weight (Mr) 4.5–6 ×106) is one of the largest well defined assemblies of proteins known, comprising multiple copies of three different enzymes10,11. The substrate is carried in thioester linkage by lipoyl–lysine residues of the lipoate acetyltransferase component, the structural core of the complex. The lipoyl–lysine residues act as swinging arms, carrying substrate between the catalytic centres of the three enzymes12–15 and between lipoic acid residues attached to different subunits in the lipoate acetyltransferase core16–18. It has been conjectured that the lipoic acid-containing regions of polypeptide chain might be flexible19,20 and therefore able to increase greatly the effective radius of a swinging arm19. We report here unexpectedly sharp lines in the 270-MHz proton NMR spectrum of the enzyme complex that are attributed to remarkable conformational mobility of large regions of polypeptide chain carrying the lipoic acid residues. This mobility would enhance the functional connection of active sites in a multisubunit structure.