Nucleotide Exchange from the High-Affinity ATP-Binding Site in SecA Is the Rate-Limiting Step in the ATPase Cycle of the Soluble Enzyme and Occurs through a Specialized Conformational State

Abstract

We have characterized the kinetic and thermodynamic consequences of adenine nucleotide interaction with the low-affinity and high-affinity nucleotide-binding sites in free SecA. ATP binds to the hydrolytically active high-affinity site approximately 3-fold more slowly than ADP when SecA is in its conformational ground state, suggesting that ATP binding probably occurs when the enzyme is in another conformational state during the productive ATPase/transport cycle. The steady-state ATP hydrolysis rate is equivalent to the rate of ADP release from the high-affinity site under a number of conditions, indicating that this process is the rate-limiting step in the ATPase cycle of the free enzyme. Because efficient protein translocation requires at least a 100-fold acceleration in the ATPase rate, the rate-limiting process of ADP release from the high-affinity site is likely to play a controlling role in the conformational reaction cycle of SecA. This release process involves a large enthalpy of activation, suggesting that it involves a protein conformational change, and two observations indicate that this conformational change is different from the well-characterized endothermic conformational transition believed to gate the binding of SecA to SecYEG. First, nucleotide binding to the low-affinity site strongly inhibits the endothermic transition but does not reduce the rate of ADP release. Second, removal of Mg(2+) from an allosteric binding site on SecA does not perturb the endothermic transition but produces a 10-fold acceleration in the rate of ADP release. These divergent effects suggest that a specialized conformational transition mediates the rate-limiting ADP-release process in SecA. Finally, ADP, 2'-O-(N-methylanthraniloyl)-adenosine-5'-diphosphate (MANT-ADP), and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) bind with similar affinities to the high-affinity site and also to the low-affinity site as inferred from their consistent effects in inhibiting the endothermic transition. In contrast, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) shows 100-fold weaker affinity than ADP for the high-affinity site and no detectable interaction with the low-affinity site at concentrations up to 1 mM, suggesting that this nonhydrolyzable analogue may not be a faithful mimic of ATP in its interactions with SecA.

Role of a Conserved Glutamate Residue in the Escherichia coli SecA ATPase Mechanism

“…It is not clear yet which state of the protein, membrane-bound or membrane-free, is stabilized upon ATP binding to SecA. There is strong evidence that binding of the non-hydrolysable ATP analog, AMP-PNP, to SecA favors the membrane-bound state (11,21), but this analog has 1000-fold lower affinity for SecA than ATP, which casts doubt on whether it can serve as a faithful mimic of ATP (5).…”

“…There also appears to be a low-affinity nucleotidebinding site on SecA, although it is not known whether this site is catalytically active and how it might contribute to the protein translocation mechanism (5-10). The high-affinity nucleotidebinding site is better defined, but even its exact role in protein translocation is not completely clear (5,(11)(12)(13)(14)(15). SecA has a low endogenous ATPase activity that is stimulated 5-10-fold by the presence of SecYEG translocon/membrane and pre-protein, highlighting the link between the ATPase and protein translocation activities (16 -18).…”

“…One interpretation of this result is that ATP binding to SecA triggers its insertion into the translocon. However, because the interaction of AMP-PNP with SecA is ϳ1000-fold weaker than that of ATP, it is not entirely clear whether this nucleotide analog can accurately mimic the effects of ATP binding to SecA (5). Thus, the question of what role ATP binding to SecA plays in the protein translocation mechanism remains open.…”

“…Thus, the question of what role ATP binding to SecA plays in the protein translocation mechanism remains open. Another notable finding is that ADP binding to the high-affinity site stabilizes a compact conformation of SecA (ground state) that has low affinity for the SecYEG/membrane (5,8,12,23). This result suggests that following ATP hydrolysis, the ADP-bound SecA undergoes retraction from the translocon to complete one reaction cycle.…”

“…This result suggests that following ATP hydrolysis, the ADP-bound SecA undergoes retraction from the translocon to complete one reaction cycle. However, the apo (nucleotide free) form of SecA can also exist in a compact conformation with low affinity for the translocon (5,24). Moreover, ADP release from SecA is stimulated by SecYEG/membrane, raising the question whether SecA retraction from the membrane occurs in the ADP-bound form, the apo form, or both (16).…”