Probes of inhibition of Escherichia coli F(1)-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.
Binding of MgADP and MgATP to Escherichia coli F(1)-ATPase (EcF(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(d) 1.3 microM) promotes the inhibition, whereas at higher concentrations (K(d) 0.7 mM) EcF(1) is protected from inhibition. The mutant betaY331W-EcF(1) requires much higher MgADP, K(d) of about 10 mM, for protection. Such MgADP binding was not revealed by fluorescence quenching measurements. MgATP partially protects EcF(1) 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(1) from inhibition by NBD-Cl. A complete protection of EcF(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(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.
Bulygin
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VV
Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA.
Milgrom
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YM
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D015335:Molecular Probes; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D015335:Molecular Probes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan