title: 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.
abstract: MgADP and MgATP binding to catalytic sites of βY341W-α(3)β(3)γ subcomplex of F(1)-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-α(3)β(3)γ 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-α(3)β(3)γ 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-α(3)β(3)γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α(3)β(3)γ 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 F(1)-ATPases.
authors: Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA. milgromy@upstate.edu
authors: Y M
authors: YM
authors: Milgrom
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001407:Bacillus / Q000201:enzymology*; D025243:Bacterial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004123:Dimethylamines / Q000737:chemistry; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism; D009578:Nitrobenzenes / Q000737:chemistry; D010080:Oxazoles / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry
chemical_list: C511330:7-chloro-4-nitrobenz-2-oxa-1,3-diazole; D004123:Dimethylamines; D050505:Mutant Proteins; D009578:Nitrobenzenes; D010080:Oxazoles; D021122:Protein Subunits; C014518:dodecyldimethylamine oxide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases