Characteristics of protection by MgADP and MgATP of [alpha]^sub 3^[beta]^sub 3^[Gamma] subcomplex of thermophilic Bacillus PS3 [beta]Y341W-mutant F^sub 1^-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a Bi-site mechanism of catalysis

• Published in: Biochemistry (Moscow) Vol. 76; no. 11; p. 1253
• Main Author: Milgrom, Y M
• Format: Journal Article
• Language: English
• Published: New York Springer Nature B.V 01.11.2011
• Subjects: Bacteria; Biochemistry; Catalysis; Enzymes; Molecular biology
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297911110071

MgADP and MgATP binding to catalytic sites of βY341W-α^sub 3^β^sub 3^Γ subcomplex of F^sub 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^sub 3^β^sub 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^sub 3^β^sub 3^Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K ^sub 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^sub 3^β^sub 3^Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α^sub 3^β^sub 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^sub 1^-ATPases.[PUBLICATION ABSTRACT]