Received January 21, 2011; Revision received April 5, 2011
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 subcomplex 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 Kd 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.
KEY WORDS: ATP synthase, catalytic cooperativity, bi-site catalysis, multi-site catalysis, nucleotide binding