Probes of inhibition of Escherichia coli F1-ATPase by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in the presence of MgADP and MgATP support a bi-site mechanism of ATP hydrolysis by the enzyme

摘要

Binding of MgADP and MgATP to Escherichia coli F^sub 1^-ATPase (EcF^sub 1^) has been assessed by their effects on extent of the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). MgADP at low concentrations (K ^sub d^ 1.3 μM) promotes the inhibition, whereas at higher concentrations (K ^sub d^ 0.7 mM) EcF^sub 1^ is protected from inhibition. The mutant βY331W-EcF^sub 1^ requires much higher MgADP, K ^sub d^ of about 10 mM, for protection. Such MgADP binding was not revealed by fluorescence quenching measurements. MgATP partially protects EcF1 from inactivation by NBD-Cl, but the enzyme remains sensitive to NBD-Cl in the presence of MgATP at concentrations as high as 10 mM. The activating anion selenite in the absence of MgATP partially protects EcF^sub 1^ from inhibition by NBD-Cl. A complete protection of EcF^sub 1^ from inhibition by NBD-Cl has been observed in the presence of both MgATP and selenite. The results support a bi-site catalytic mechanism for MgATP hydrolysis by F^sub 1^-ATPases and suggest that stimulation of the enzyme activity by activating anions is due to the anion binding to a catalytic site that remains unoccupied at saturating substrate concentration.[PUBLICATION ABSTRACT]