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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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Abstract

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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Abbreviations

F1 :

solubilized portion of FoF1-ATP synthase

MF1, EcF1, and TF1 :

F1-ATPases from beef-heart mitochondria, Escherichia coli, and thermophilic Bacillus PS3, respectively

NBD-Cl:

7-chloro-4-nitrobenz-2-oxa-1,3-diazole

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  1. Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York, 13210, USA

    V. V. Bulygin & Y. M. Milgrom

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  1. V. V. Bulygin
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Correspondence to Y. M. Milgrom.

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Published in Russian in Biokhimiya, 2010, Vol. 75, No. 3, pp. 400–410.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM09-235, January 17, 2010.

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Bulygin, V.V., Milgrom, Y.M. 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. Biochemistry Moscow 75, 327–335 (2010). https://doi.org/10.1134/S0006297910030090

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