Singh S K, Maurizi M R
Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Nov 25;269(47):29537-45.
ClpA, the regulatory subunit of Clp protease from Escherichia coli, has two ATP-binding sites in non-homologous regions of the protein, referred to as domain I and domain II. We have mutated the invariant lysine in the ATP-binding sites of domain I and domain II and studied the enzymatic properties of the purified mutant ClpA proteins. The domain I mutant, ClpA-K220Q, was unable to form a hexamer in the presence of nucleotide, but the comparable domain II mutant, ClpA-K501Q, associated into a hexamer in the presence of ATP, indicating that nucleotide binding to domain I favors a conformation required to stabilize the quaternary structure of ClpA. ClpA-K220Q was defective in ATPase activity and in the ability to activate protein and peptide degradation by ClpP, but the defects could be partially overcome by formation of hybrid hexamers with wild-type ClpA. Another domain I mutant, ClpA-K220R, readily formed hexamers in the presence of ATP and retained > or = 60% of the wild-type ATPase activity and ability to activate ClpP. These results indicate that hexamer formation is a prerequisite for expression of enzymatic activity. Domain II mutants ClpA-K501Q and ClpA-K501R had very low ATPase activity (< 10% of wild-type) and a severe defect in activation of protein degradation, which requires ATP hydrolysis. Domain II mutants were able to activate ClpP to degrade a peptide whose degradation required nucleotide binding but not hydrolysis. Nucleotide binding to domain II of ClpA is important to form a productive complex with ClpP, and domain II appears to be primarily responsible for an energy-requiring step in the catalytic cycle unique to the degradation of large proteins.
ClpA是来自大肠杆菌的Clp蛋白酶的调节亚基，在该蛋白质的非同源区域有两个ATP结合位点，分别称为结构域I和结构域II。我们对结构域I和结构域II的ATP结合位点中的不变赖氨酸进行了突变，并研究了纯化的突变型ClpA蛋白的酶学性质。结构域I突变体ClpA-K220Q在核苷酸存在的情况下无法形成六聚体，但与之类似的结构域II突变体ClpA-K501Q在ATP存在时能组装成六聚体，这表明核苷酸与结构域I的结合有利于稳定ClpA四级结构所需的构象。ClpA-K220Q在ATP酶活性以及激活ClpP介导的蛋白质和肽降解的能力方面存在缺陷，但通过与野生型ClpA形成杂合六聚体，这些缺陷可以得到部分克服。另一个结构域I突变体ClpA-K220R在ATP存在时很容易形成六聚体，并保留了≥60%的野生型ATP酶活性和激活ClpP的能力。这些结果表明，六聚体的形成是酶活性表达的前提条件。结构域II突变体ClpA-K501Q和ClpA-K501R的ATP酶活性非常低（<野生型的10%），并且在激活蛋白质降解方面存在严重缺陷，而蛋白质降解需要ATP水解。结构域II突变体能够激活ClpP降解一种其降解需要核苷酸结合但不需要水解作用的肽。核苷酸与ClpA的结构域II结合对于与ClpP形成有活性的复合物很重要，并且结构域II似乎主要负责在催化循环中降解大蛋白质所特有的一个需要能量的步骤。
