BIOCHEMISTRY OF ENERGY-DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

ATP依赖性蛋白酶在翻译后蛋白质合成中起关键作用。 调节重要细胞蛋白质的数量和活性 以及从细胞中去除受损和变性的蛋白质。我们 研究集中在ATP的生物化学特征上， 依赖性Clp和Lon蛋白酶。大肠杆菌和新发现的 ATP依赖性蛋白酶。两个ATP酶位点的定点突变 在ClpA中的研究表明，ClpA的活性形式是六聚体， 亚基之间的相互作用影响ATP酶和蛋白酶- ClpA的激活活性。域I网站被证明参与 ClpA六聚体的组装以及与ClpP和结构域II的相互作用 需要一个位点来激活ClpP的蛋白水解活性， 大蛋白质模型肽底物已被合成并显示 比以前的任何衬底都更快地被切割。这些肽 与ClpA紧密结合，激活ATP酶活性。现场 在由肽占据的ClpA上定义了蛋白质的变构位点 在酶上，这个位点的占据似乎是打开所必需的。 大多肽的ClpP活性位点，并加速 肽键的催化裂解。使用部分灭活的ClpP， 证明了多肽和蛋白质的进行性切割 需要以十二聚体形式存在的活性位点阵列， ClpP。当非酶切时，观察到模型肽的过程性切割。 ATP的可水解类似物用于激活酶，表明 ATP水解在持续合成能力中不起直接作用。研究 与Susan Gottesman博士合作完成的研究表明， 另一个ATP酶来自E. coli，ClpX，与ClpA进化相关， 这种蛋白质在体内与ClpP一起发挥作用， 一种降解高度不稳定的λ O蛋白的蛋白水解系统。 序列和生物化学数据现在表明在E.大肠杆菌四 ATP酶、Clps A、B、X和Y的变化表明， 即使是这种简单生物体的蛋白水解系统也是高度复杂的。在 与Michael Gottesman博士和Nan Wang博士合作完成的研究， 已发现ATP依赖性Lon蛋白酶的一种密切同源物 发生在人类线粒体中。进一步的生物化学和免疫化学 研究正在进行中，以确定这种物质的性质和功能。 蛋白酶的作用。

ATP-dependent proteases play critical roles in the post-translational regulation of the amounts and activities of important cellular proteins and in the removal of damaged and denatured proteins from the cell. Our research has focussed on the biochemical characterization of the ATP- dependent Clp and Lon proteases from E. coli and on the discovery of new ATP-dependent proteases. Site-directed mutagenesis of the two ATPase sites in ClpA demonstrated that the active form of ClpA is a hexamer and that interactions between the subunits affect the ATPase and protease- activating activity of ClpA. The domain I site was shown to be involved in assembly of the ClpA hexamer, and interaction with ClpP and the domain II site was needed for activation of the proteolytic activity ClpP against large proteins. Model peptide substrates have been synthesized and shown to be cleaved more rapidly than any previous substrates. These peptides interacted tightly with ClpA and activated its ATPase activity. The site on ClpA occupied by the peptides defines the allosteric site for proteins on the enzyme, and occupancy of this site appears to be necessary to open the active site of ClpP for large polypeptides and to accelerate the catalytic cleavage of peptide bonds. Using partially inactivated ClpP, it was demonstrated that processive cleavage of polypeptides and proteins required the array of active sites present in the dodecameric form of ClpP. Processive cleavage of model peptides was observed when non- hydrolyzable analogs of ATP were used to activate the enzyme, indicating that ATP hydrolysis does not play a direct role in processivity. Studies done in collaboration with Dr. Susan Gottesman have demonstrated that another ATPase from E. coli, ClpX, is evolutionarily related to ClpA and that this protein functions in vivo with ClpP as an essential component of a proteolytic system that degrades the highly unstable lambda O protein. Sequence and biochemical data now indicate the presence in E. coli of four ATPases, Clps A, B, X and Y and suggest that the energy-dependent proteolytic systems of even this simple organism are highly complex. In studies done in collaboration with Drs. Michael Gottesman and Nan Wang, it has been found that a close homolog of the ATP-dependent Lon protease occurs in human mitochondria. Further biochemical and immunochemical studies are underway to identify the properties and function of this protease in human cells.