CHEMICAL SYNTHESIS OF CYTOCHROME C--THE ROLES OF INDIVIDUAL RESIDUES

目前物种的线粒体细胞色素c被推测为 共同反映了过去15亿年发生的进化事件 好几年了。这些进化秘密，如果通过寻找角色而被揭开 不变残基，可能有助于理解其背后的原理 蛋白质的结构与功能。在这种情况下，有必要 化学合成W.M.预测的原始形式的细胞色素c。 Fitch和E.Martgoliash并测试其是否处于活动状态。的可行性。 这个项目得到了我们的发现的支持，即互补片段 在马和克鲁斯假丝酵母细胞色素c之间完全可交换。 形成杂化络合物(见另一份报告)。作为第一步， 我们计划通过合成细胞色素c来合成马的细胞色素c， 用Merrifield固相法连接两个分支 (络合辅助重新加入，见上一份报告)，随后 使用在本发明中发现的细胞色素c合成酶，对血红素进行酶促附着 一节。在延长加入程序的过程中，我们已经表明， (HSE-内酯-65)(23-65)(高丝氨酸-内酯)，但不 (HSE-内酯-65)(39-65)，马细胞色素c与阿夫林重新结合 (66-104)在还原的血红素片段(1-25)H存在和完整的情况下 蛋氨酸80对这种重合反应是必不可少的。在…的基础上 这些结果，使半合成(HSE-65)(28-104)，我们进行了 马细胞色素c片段(Gly-66)(28-66)的合成 色氨酸在第59位。 在努力开发第二种片段连接程序的过程中，我们 发现了一种新的羧基肽酶A催化的转肽，如下所示。 合成放射性标记三肽Leu-Met-Het-酰胺或Leu-Arg-Met-酰胺 和过量的酵母细胞色素c的(HSE-69)(1-69)H或 马细胞色素c的(HSE-65)(1-65)H与羧肽酶孵育 A在pH为10.0、20-30℃、有50-70%甘油存在的情况下。 对产物的分析表明，两个标记的三肽都被替换了 羧基末端高丝氨酸，7天后效率可达50%。

The mitochondrial cytochromes c of the present species are presumed to collectively reflect evolutionary events occurring for the last 1.5 billion years. These evolutionary secrets, if uncovered through finding the roles of the invariant residues, may help understanding the principles underlying structure-function of proteins. In this context, it would be worthwhile to chemically synthesize the ancestral form of cytochrome c predicted by W.M. Fitch and E. Martgoliash and test whether it is active. The feasibility of this project is supported by our finding that the complementing fragments are completely exchangeable between horse and Candida krusei cytochromes c for formation of hybrid complexes (see the other report). As a first step, we plan to synthesize horse cytochrome c via synthesis of apocytochrome c, using the Merrifield solid phase method and joining two apofragments (complexation assisted rejoining, see the previous report), followed enzymatic attachment of heme, using cytochrome c synthase found in this Section. In extending the joining procedure we have shown that apofragment (Hse-lactone-65)(23-65) (homoserine-lactone), but not (Hse-lactone-65)(39-65), of horse cytochrome c rejoins to apofragment (66-104) in the presence of reduced heme fragment (1-25)H and that intact methionine 80 is essential for this rejoining reaction. On the basis of these results, to make semi-synthetic (Hse-65)(28-104), we carried out synthesis of fragment (Gly-66)(28-66) of horse cytochrome c, containing tryptophan at position 59. In the effort to develop a second procedure for joining of fragments, we found a novel carboxypeptidase A catalyzed transpeptidation as follows. Synthesized radiolabeled tripeptide Leu-Met-His-amide or Leu-Arg-Met-amide and excess of heme fragment (Hse-69)(1-69)H of yeast cytochrome c or (Hse-65)(1-65)H of horse cytochrome c were incubated with carboxypeptidase A in the presence of 50 to 70% glycerol at pH 10.0 at 20 to 30 degrees C. Analyses of the product indicated that both labeled tripeptides replaced the carboxyterminal homoserine with the efficiency up to 50% after 7 days.