Two independent mechanisms down-regulate the intrinsic SecA ATPase activity.

SecA initiates protein translocation by interacting with ATP, preprotein, and the SecYEG membrane components. Under such conditions, it undergoes a conformational change characterized as membrane insertion, which is then followed by hydrolysis of ATP, enabling the release of the preprotein and deinsertion of SecA itself for the next cycle of reactions. Without ongoing translocation, the ATPase activity of SecA is kept very low. Previously, it was shown that the C-terminal 34-kDa domain of SecA interacts with the N-terminal 68-kDa ATPase domain to down-regulate the ATPase. Here, we show, using a deregulated SecA mutant, that the intrinsic ATPase activity is subject to dual inhibitory mechanisms. Thus, the proposed second ATP-binding domain down-regulates the ATPase activity executed by the primary ATPase domain. This regulation, within the N-terminal ATPase domain, operates independently of the C-terminal domain-mediated regulation. The absence of both the mechanisms resulted in a 50-fold elevation of translocation-uncoupled ATP hydrolysis.