BIOCHEMISTRY OF ENERGY-DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

依赖于ATP的蛋白水解酶负责很大一部分 真核和原核细胞内蛋白质的降解 细胞。大肠杆菌CLP蛋白水解酶是一个普遍家族的代表 由一个蛋白分解核心(ClpP)组成的依赖于ATP的蛋白酶 和几个依赖于ATP的调节亚单位(ClpA-家族成员)。 调节亚基和蛋白水解亚单位之间的相互作用 可能影响体内CLP蛋白水解酶的特异性和活性。 我们的研究集中在大肠杆菌CLP蛋白酶的生物化学上。 目的是(1)确定蛋白酶的专一性和 它在体内选择靶点的机制，以及(2)定义 ATP在蛋白质选择和降解中的作用。我们有 成功地明确定义了ATP的两个角色。ATP充当一种 变构效应器用于组装配合物之间的 调节成分ClpA和蛋白分解成分ClpP。 ClpA和ClpP之间的相互作用反过来改变了ClpP的活性部位， 通过切割中长肽的能力来表示的。 然而，大型蛋白质的降解需要ATP水解， 表明降解的第二步需要三磷酸腺苷。这个 依赖于ATP水解的步骤可能涉及到改变 大蛋白底物的结构(伴侣功能)或改变 蛋白质底物与酶的相互作用 (易位功能)。ClpA与蛋白质的直接相互作用 而这些底物对ATPase的影响表明了多肽的存在 在没有ClpP的情况下，ClpA的活性。蛋白水解酶的选择性 CLP应同时涉及活性和变构的相互作用 位点，因为模型肽底物的降解表明活性 ClpP的部位具有较广泛的特异性。基因的定点突变 ClpA结构域2中的ATPase位点表明在该位置上的水解度 活性CLP的组装和切割不需要位点 中长肽。因为在区域2中改变的突变体不能 降解大的蛋白质，该结构域似乎负责 中电在降解过程中的步骤。

ATP-dependent proteases are responsible for a major portion of the degradation of intracellular proteins in eukaryotic and prokaryotic cells. The E. coli Clp protease is representative of a universal family of ATP-dependent proteases that are composed of a proteolytic core (ClpP) and several ATP-dependent regulatory subunits (ClpA-family members). Interactions between the regulatory subunits and the proteolytic subunit may affect the specificity and activity of the Clp proteases in vivo. Our research has focused on the biochemistry of E. coli Clp protease with the aims of (1) defining the specificity of the protease and the mechanism by which it selects targets in vivo, and (2) defining the functions of ATP in the selection and degradation of proteins. We have succeeded in clearly defining two roles for ATP. ATP acts as an allosteric effector for the assembly of the complex between the regulatory component, ClpA, and the proteolytic component, ClpP. Interaction between ClpA and ClpP in turn alters the active site of ClpP, indicated by the ability to cleave intermediate length peptides. Degradation of large proteins, however, requires ATP hydrolysis, indicating that ATP is required for a second step in degradation. The ATPhydrolysis-dependent step probably involves alterations in the structure of large protein substrates (chaperone function) or changes in the interactions between the protein substrates and the enzyme (translocation function). Direct interaction between ClpA and proteins and peptides is indicated by the effects of these substrates on ATPase activity of ClpA in the absence of ClpP. Selectivity of proteolysis by Clp should involve interactions at both the active and the allosteric sites, since degradation of model peptide substrates indicates the active site of ClpP has rather broad specificity. Site-directed mutagenesis of the ATPase site in domain 2 of ClpA indicates that hydrolysis at that site is not required for assembly of active Clp and cleavage of intermediate length peptides. Since mutants altered in domain 2 cannot degrade large proteins, that domain appears to be responsible for the processive steps in degradation by Clp.