Radford S E, Laue E D, Perham R N, Miles J S, Guest J R
Department of Biochemistry, University of Cambridge, U.K.
Biochem J. 1987 Nov 1;247(3):641-9. doi: 10.1042/bj2470641.
A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain. An active complex is still formed and 1H-n.m.r. spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared. A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain. 1H-n.m.r. spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum. These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain. Closer examination of the 400 MHz 1H-n.m.r. spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance. This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain. If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile. The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.
可以在编码大肠杆菌丙酮酸脱氢酶多酶复合体的aceEF-lpd操纵子中进行体外缺失操作，这会导致构成每个二氢硫辛酰胺乙酰转移酶（E2p）多肽链N端一半的三个同源硫辛酰结构域中的两个缺失。仍然会形成活性复合体，对这种修饰后的复合体进行的1H核磁共振光谱分析表明，许多先前归因于野生型E2p多肽链中构象可移动片段的异常尖锐共振相应消失了。在将剩余的一个硫辛酰结构域与E2p链的C端一半连接起来的富含（丙氨酸 + 脯氨酸）的长多肽链片段中设计了进一步的缺失。对所得同样具有活性的酶复合体进行的1H核磁共振光谱分析表明，光谱中观察到的异常尖锐共振进一步相应减少。这些实验有力地支持了这样一种观点，即尖锐共振至少主要源自分隔三个硫辛酰结构域并将它们与E2p链的C端一半连接起来的三个富含（丙氨酸 + 脯氨酸）的长序列。对野生型和重组复合体以及有限蛋白酶解产物的400 MHz 1H核磁共振光谱进行更仔细的检查，发现了另一个尖锐但较小的共振。这初步归因于另一个但较小的富含（丙氨酸 + 脯氨酸）的序列，该序列将二氢硫辛酰胺脱氢酶结合结构域与E2p链C端一半中的内核结构域分隔开。如果这个序列在构象上也是灵活的，那么它可能解释了先前的荧光数据，这些数据表明与酶复合体结合的二氢硫辛酰胺脱氢酶相当可移动。E2p链中的乙酰转移酶活性位点位于内核结构域中，在残基370和629之间。
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