AMINO ACID METABOLISM--ENZYME BIOGENESIS AND MUTATION

在本申请中提出的研究持续了很长时间 对线粒体核编码机制的兴趣 蛋白质被靶向、转位、转位后 加工、折叠并组装成其本机的、活动的 结构。这些过程是维护和 线粒体的繁殖，因此对细胞新陈代谢和 动态平衡。彻底了解和理解这些 通路是阐明人类致病机制的关键 涉及线粒体酶的代谢性疾病和 合理设计其躯体矫治策略 不足之处。用于这些研究的模型系统是完整的大鼠 肝脏线粒体及其亚组分或组分的纯化 由此，模型底物为哺乳动物鸟氨酸 转氨甲基酶(OTC)，一种肝脏特异的线粒体酶 缺乏高氨血症的。具体目标包括：i) 线粒体加工肽酶(MPP)的特性， 负责转位后的一种酶 通过在大肠杆菌中过表达来处理OTC，定义其 活性部位和金属离子要求，通过以下方式扰乱其活动 定点突变、结晶和解决其 结构；2)识别、提纯和表征其他 切割进口部分线粒体所需的蛋白水解酶 前体；3)分离和克隆先导肽受体和 移位装置的部件(接触部位)由 恢复易位复合体，交联易位 中间体到仪器的成员，并提纯鉴定 蛋白质免疫化学；4)探索个体的作用 伴侣蛋白GroEL中的氨基酸残基(与大鼠同源 HSP60蛋白)参与ATP水解酶、底物蛋白结合 激活和蛋白质折叠；以及5)确定 多聚体酶或复合体是一种蛋白质。经调解的 过程以及所需因素的性质和一致性是什么 用于此功能。

The research proposed in this application continues a long-standing interest in the mechanisms by which nuclearly coded mitochondrial proteins are targeted, translocated, posttranslocationally processed, folded, and assembled into their native, active structures. These processes are central to the maintenance and propagation of mitochondria, and, hence, to cellular metabolism and homeostasis. A thorough knowledge and understanding of these pathways is critical to illuminating the pathogenesis of human metabolic disease involving mitochondrial enzymes and to the rational design of strategies for somatic correction of their deficiencies. The model system used for these studies is intact rat liver mitochondria and subfractions or components purified therefrom, and the model substrate is mammalian ornithine transcarbamylase (OTC), a liver-specific, mitochondrial enzyme deficient in hyperammonemia. The specific aims include: i) characterization of the mitochondria processing peptidase (MPP), one of the enzymes responsible for the posttranslocational processing of OTC by overexpressing it in E. coli, defining its active site and metal ion requirements, disrupting its activity by site-directed mutagenesis, crystallizing it, and solving its structure; 2) identifying, purifying, and characterizing other mitochondrial proteases required to cleave a subset of imported precursors; 3) isolating and cloning leader peptide receptors and components of the translocation apparatus (contact site) by recovering translocation complexes, cross-linking translocation intermediates to members of the apparatus, and purifying identified proteins immunochemically; 4) exploring the role of individual amino acid residues in the chaperonin groEL (homologous to the rat Hsp60 protein) in ATP hydrolysis, substrate protein binding, groES activation, and protein folding; and 5) determining whether multimeric enzyme or complex assembly is a protein. mediated process and what is the nature and identity of the factors required for this function.