The motor protein SecA drives the transport of polypeptides through the ubiquitous protein channel SecYEG. Changes in protein-nucleotide binding energy during the hydrolytic cycle of SecA must be harnessed to drive large conformational changes resulting in channel opening and vectorial substrate polypeptide transport. Here, we elucidate the ATP hydrolysis cycle of SecA from Escherichia coli by transient and steady-state methods. The basal ATPase activity of SecA is very slow with the release of ADP being some 600-fold slower than hydrolysis. Upon binding to SecYEG the release of ADP is stimulated but remains rate-limiting. ADP release is fastest in the fully coupled system when a substrate protein is being translocated; in this case hydrolysis and ADP release occur at approximately the same rate. The data imply that ADP dissociation from SecA is accompanied by a structural rearrangement that is strongly coupled to the protein interface and protein translocation through SecYEG.ATPase ͉ SecYEG ͉ steady-state kinetics ͉ transient kinetics ͉ translocon A TP-dependent molecular motors couple hydrolytic power to mechanical movement in a controlled and directional fashion. Distortions in the structure of the active site driven by different chemical stages in the ATP hydrolysis cycle can be relayed across large distances, resulting in conformational changes in the ATPase and its interacting partners. Hence, ATP binding, formation of the initial ADP:Pi complex or release of either of the products can be coupled to different motions required to drive the protein machine. The reverse can also be accomplished in the capture of free energy e.g., by the F 1 F 0 -ATP synthase (1, 2).The subject of this study, SecA, is an ATPase and the active component of the bacterial protein translocation machinery that also contains the protein channel SecYEG (3, 4). It peripherally associates with the membrane, receives unfolded secretory proteins and facilitates their passage across the inner membrane (5, 6). The structure of SecA reveals a single nucleotide binding site situated between 2 RecA-like folds, a scaffold, wing and preprotein cross-linking domain (7).Addition to SecA of preprotein and SecYEG reconstituted into an acidic phospholipid membrane promotes protein translocation and an increase in the rate of ATP hydrolysis (4,8). This corresponds to the energy-transducing activity and is a multistep process (9-11). How the structure of SecA or the channel (12, 13) relate to specific stages of the ATP coupled reaction is unclear.Although there is still controversy in the field as to the nature and purpose of homo-oligomerisation in SecA, an increasing body of evidence suggests that SecA is dimeric in free solution, but dissociates into monomers as a consequence of the protein transport reaction (14-21). Additional rearrangements within SecA (14,19,21,22) and SecYEG (21,23,24) have also been reported. These studies have been crowned by an X-ray structure of the complex (one copy of each) that reveals the nature and consequences of the...

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