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

• Published in: Biochemistry (Moscow) 2011-11, Vol.76 (11), p.1253-1261
• Main Author: Milgrom, Y. M.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Triphosphate - chemistry; Bacillus - enzymology; Bacterial Proton-Translocating ATPases - antagonists & inhibitors; Bacterial Proton-Translocating ATPases - chemistry; Binding Sites; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Catalysis; Catalytic Domain; Dimethylamines - chemistry; Kinetics; Life Sciences; Microbiology; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Nitrobenzenes - chemistry; Oxazoles - chemistry; Protein Subunits - chemistry
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297911110071

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 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-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.