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Characteristics of protection by MgADP and MgATP of α3β3Γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a Bi-site mechanism of catalysis

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Abstract

MgADP and MgATP binding to catalytic sites of βY341W-α3β3Γ subcomplex of F1-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α3β3Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α3β3Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α3β3Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α3β3Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F1-ATPases.

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Abbreviations

BSA:

bovine serum albumin

F1 :

the solubilized portion of FoF1-ATP synthase

LDAO:

lauryldimethylamine-N-oxide

MF1, EcF1, and TF1, F1 :

ATPases from mitochondria Escherichia coli, and thermophilic Bacillus PS3, respectively

NBD-Cl:

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

PEP:

phosphoenolpyruvate

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Authors and Affiliations

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

    Y. M. Milgrom

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Correspondence to Y. M. Milgrom.

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Published in Russian in Biokhimiya, 2011, Vol. 76, No. 11, pp. 1556–1565.

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Milgrom, Y.M. Characteristics of protection by MgADP and MgATP of α3β3Γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a Bi-site mechanism of catalysis. Biochemistry Moscow 76, 1253–1261 (2011). https://doi.org/10.1134/S0006297911110071

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  • DOI: https://doi.org/10.1134/S0006297911110071

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