van der Wolk J P, de Wit J G, Driessen A J
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
EMBO J. 1997 Dec 15;16(24):7297-304. doi: 10.1093/emboj/16.24.7297.
SecA is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade, and is bound at the membrane surface to the integral membrane domain of the preprotein translocase. Preproteins are thought to be translocated in a stepwise manner by nucleotide-dependent cycles of SecA membrane insertion and de-insertion, or as large polypeptide segments by the protonmotive force (Deltap) in the absence of SecA. To determine the step size of a complete ATP- and SecA-dependent catalytic cycle, translocation intermediates of the preprotein proOmpA were generated at limiting SecA translocation ATPase activity. Distinct intermediates were formed, spaced by intervals of approximately 5 kDa. Inhibition of the SecA ATPase by azide trapped SecA in a membrane-inserted state and shifted the step size to 2-2.5 kDa. The latter corresponds to the translocation elicited by binding of non-hydrolysable ATP analogues to SecA, or by the re-binding of partially translocated polypeptide chains by SecA. Therefore, a complete catalytic cycle of the preprotein translocase permits the stepwise translocation of 5 kDa polypeptide segments by two consecutive events, i.e. approximately 2.5 kDa upon binding of the polypeptide by SecA, and another 2.5 kDa upon binding of ATP to SecA.
SecA是大肠杆菌前体蛋白转运级联反应中依赖ATP的驱动力产生器，它在膜表面与前蛋白转位酶的整合膜结构域结合。前蛋白被认为是通过SecA膜插入和去插入的核苷酸依赖性循环以逐步方式转运，或者在没有SecA的情况下通过质子动力（Δp）以大的多肽片段形式转运。为了确定完整的依赖ATP和SecA的催化循环的步长，在前体蛋白proOmpA的SecA转运ATP酶活性受限的情况下产生了转运中间体。形成了不同的中间体，间隔约为5 kDa。叠氮化物对SecA ATP酶的抑制作用将SecA捕获在膜插入状态，并将步长转移到2 - 2.5 kDa。后者对应于不可水解的ATP类似物与SecA结合引发的转运，或SecA对部分转运的多肽链的重新结合引发的转运。因此，前蛋白转位酶的完整催化循环通过两个连续事件允许5 kDa多肽片段的逐步转运，即SecA结合多肽时约2.5 kDa，以及ATP结合到SecA时再转运2.5 kDa。
