Scopul nostru este sprijinirea şi promovarea cercetării ştiinţifice şi facilitarea comunicării între cercetătorii români din întreaga lume.
Autori: John J. Fak, Anna Itkin, Daita D. Ciobanu, Edward C. Lin, Xiang-Jin Song, Yi‑Te Chou, Lila M. Gierasch, and John F. Hunt
Editorial: Biochemistry, 43(23);, p.7307-7327, 2004.
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 Mg2+ 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 ratelimiting
ADP-release process in SecA. Finally, ADP, 2¢-O-(N-methylanthraniloyl)-adenosine-5¢-diphosphate
(MANT-ADP), and adenosine 5¢-O-(3-thiotriphosphate) (ATP-ç-S) bind with similar affinities to the highaffinity
site and also to the low-affinity site as inferred from their consistent effects in inhibiting the
endothermic transition. In contrast, adenosine 5¢-(â,ç-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.
Cuvinte cheie: SecA; ATPase; protein translocation; MANT-ADP; kinetics; fluorescence anisotropy.