title: ATP synthesis by F0F1-ATP synthase independent of noncatalytic nucleotide binding sites and insensitive to azide inhibition.
abstract: ATP hydrolyzing activity of a mutant alpha3beta3gamma subcomplex of F0F1-ATP synthase (DeltaNC) from the thermophilic Bacillus PS3, which lacked noncatalytic nucleotide binding sites, was inactivated completely soon after starting the reaction (Matsui, T., Muneyuki, E. , Honda, M., Allison, W. S., Dou, C., and Yoshida, M. (1997) J. Biol. Chem. 272, 8215-8221). This inactivation is caused by rapid accumulation of the "MgADP inhibited form" which, in the case of wild-type enzyme, would be relieved by ATP binding to noncatalytic sites. We reconstituted F0F1-ATP synthase into liposomes together with bacteriorhodopsin and measured illumination-driven ATP synthesis. Remarkably, DeltaNC F0F1-ATP synthase catalyzed continuous turnover of ATP synthesis while it could not promote ATP-driven proton translocation. ATP synthesis by DeltaNC F0F1-ATP synthase, as well as wild-type enzyme, proceeded even in the presence of azide, an inhibitor of ATP hydrolysis that stabilizes the MgADP inhibited form. The time course of ATP synthesis by DeltaNC F0F1-ATP synthase was linear, and gradual acceleration to the maximal rate, which was observed for the wild-type enzyme, was not seen. Thus, ATP synthesis can proceed without nucleotide binding to noncatalytic sites even though the rate is sub-maximal. These results indicate that the MgADP inhibited form is not produced in ATP synthesis reaction, and in this regard, ATP synthesis may not be a simple reversal of ATP hydrolysis.
authors.affiliation: Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Bald
authors.forename: T
authors.initials: T
authors.lastname: Amano
authors.forename: E
authors.initials: E
authors.lastname: Muneyuki
authors.forename: B
authors.initials: B
authors.lastname: Pitard
authors.forename: J L
authors.initials: JL
authors.lastname: Rigaud
authors.forename: J
authors.initials: J
authors.lastname: Kruip
authors.forename: T
authors.initials: T
authors.lastname: Hisabori
authors.forename: M
authors.initials: M
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Shibata
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001386:Azides / Q000494:pharmacology*; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006868:Hydrolysis; D017136:Ion Transport; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011522:Protons
chemical_list: D001386:Azides; D004791:Enzyme Inhibitors; D011522:Protons; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases