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

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V. V. Bulygin^# and Y. M. Milgrom^*

Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA; fax: +1-315-464-8750; E-mail: milgromy@upstate.edu

^# Deceased.

^* To whom correspondence should be addressed.

Received July 30, 2009; Revision received September 18, 2009

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Binding of MgADP and MgATP to Escherichia coli F₁-ATPase (EcF₁) 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 µM) promotes the inhibition, whereas at higher concentrations (K_d 0.7 mM) EcF₁ is protected from inhibition. The mutant βY331W-EcF₁ 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 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 EcF₁ from inhibition by NBD-Cl. A complete protection of EcF₁ 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 F₁-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.

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KEY WORDS: anion activation, ATP synthase, catalytic cooperativity, bi-site catalysis, nucleotide binding, tri-site catalysis

DOI: 10.1134/S0006297910030090

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