Study on structure of cytochrome b using antimycin A-resistant mutants.

为了探讨抗霉素A与其结合部位的相互作用，用新合成的含有牛心亚线粒体颗粒的抗霉素A衍生物研究了抑制作用所需的抗霉素A分子的结构因素。特别是，我们重点研究了连接水杨酸和双内酯环部分的酰胺键桥和水杨酸部分中的3-甲酰氨基的作用。由于酚羟基和酰胺羰基之间没有形成分子内氢键，导致活性显著下降(损失了四个数量级)，表明这种氢键是抑制作用的关键。这一结果表明，酚羟基和羰基都以固定构象与一些残基形成了氢键。此外，酰胺键部分的N-甲基化显著降低了抑制活性，表明NH基团可能与结合部位发生氢键相互作用。3-甲酰氨基的N-甲基化也导致了活性的降低，这可能是由于该官能团失去了旋转自由所致。对3-甲酰氨基构象的分子轨道计算研究表明，当甲酰基投射到酚羟基的相反侧时，该基团具有活性构象。在对合成的抗霉素A类似物进行一系列构效关系研究的基础上，我们提出了抗霉素A与其结合腔结合的初步模型。

The structural factors of antimycin A molecule requred for inhibitory action were studied using newly synthesized antimycin A derivatives with bovine heart submitochondrial particles, in order to probe the interaction between antimycin A and its binding site. In particular, we focussed upon the roles of the amide bond bridge, which connects the salicylic acid and dilactone-ring moieties, and the 3-formylamino group in the salicylic acid moiety. The lack of formation of an intramolecular hydrogen-bond between phenolic OH and amide carbonyl groups resulted in a remarkable loss of the activity (by four orders of magnitude), indicating that this hydrogen-bond is essential for the inhibition. This result suggested that both the phenolic OH and the carbonyl groups form a hydrogen-bond with some residues at a fixed conformation. In addition, the inhibitory potency was remarkably decreased by N-methylation of the amide bond moiety, indicating that the NH group might function in hydrogen-bond interaction with the binding site. The N-methylation of 3-formylamino group also resulted in a decrease in the activity, probably due to a loss of the rotational freedom of this functional group. Molecular orbital calculation studies with respect to the conformation of the 3-formylamino group indicated that this group takes an active conformation when the formyl carbonyl projects to the opposite side of the phenolic OH group. Based upon a series of structure-activity studies of synthetic antimycin A analogues, we propose a tentative model for antimycin A binding in its binding cavity.