Karata K, Inagawa T, Wilkinson A J, Tatsuta T, Ogura T
Department of Molecular Cell Biology, Institute of Molecular Embryology, Kumamoto University School of Medicine, Kumamoto 862-0976, Japan.
J Biol Chem. 1999 Sep 10;274(37):26225-32. doi: 10.1074/jbc.274.37.26225.
Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.
大肠杆菌FtsH是一种依赖ATP的蛋白酶，属于AAA蛋白家族。同源性第二区域（SRH）是AAA家族成员中高度保守的基序，部分地将这些蛋白质与更广泛的沃克型ATP酶家族区分开来。尽管它在AAA蛋白家族中具有保守性，但关于SRH的功能却知之甚少。为了解决这个问题，我们将点突变系统地引入FtsH的SRH中，并研究了突变蛋白的活性。发现SRH内高度保守的氨基酸残基对FtsH的功能至关重要，这些位置的突变导致ATP酶活性降低或丧失。突变对FtsH蛋白酶活性的影响与其对ATP酶活性的影响显著相关。ATP酶缺陷型SRH突变体经历了类似于野生型FtsH的ATP诱导构象变化，表明SRH在ATP水解而非ATP结合中起重要作用。根据N - 乙基马来酰亚胺敏感融合蛋白六聚化结构域的晶体结构对数据进行分析，提出了一种AAA ATP酶进行ATP水解的合理机制，该机制涉及SRH的分子间催化作用。
