BIOCHEMISTRY OF ENERGY-DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

ATP依赖性蛋白酶负责蛋白质的主要部分。 真核和原核细胞内蛋白质的降解 细胞 急诊大肠杆菌Clp蛋白酶是一个通用家族的代表 由蛋白水解核心（ClpP）组成的ATP依赖性蛋白酶 和几个ATP依赖性调节亚基（ClpA家族成员）。 调节亚基和蛋白水解亚基之间的相互作用 可能影响Clp蛋白酶在体内的特异性和活性。 我们的研究主要集中在E.大肠杆菌Clp蛋白酶， 目的是（1）确定蛋白酶的特异性， 它在体内选择靶点的机制，以及（2）定义 ATP在蛋白质的选择和降解中的作用。 我们有 成功地明确了ATP的两个角色。 ATP作为 用于组装复合物之间的变构效应物， 调节组分ClpA和蛋白水解组分ClpP。 ClpA和ClpP之间的相互作用进而改变ClpP的活性位点， 其表现为切割中等长度肽的能力。 然而，大蛋白质的降解需要ATP水解， 这表明ATP是降解的第二步所必需的。 的 ATP水解依赖性步骤可能涉及改变 大蛋白质底物的结构（分子伴侣功能）或 蛋白质底物和酶之间的相互作用 （易位功能）。 ClpA与蛋白质的直接相互作用 而多肽则通过这些底物对ATP酶的影响来指示 ClpA在ClpP不存在下的活性。 蛋白水解的选择性 CLP应该包括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位点具有较广的特异性。 的定点诱变 ClpA结构域2中ATP酶位点表明， 位点不是组装活性Clp和切割 中等长度的肽。 由于结构域2的突变体不能 降解大的蛋白质，该结构域似乎负责 Clp降解的进行性步骤。

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.