title: The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F(1)-ATPase.
abstract: F1-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P(i). Here, using a mutant alpha(3)beta(3)gamma complex of thermophilic F(1)-ATPase (alpha W463F/beta Y341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that P(i) interfered with the binding of MgATP to F(1)-ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F(1)-ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (alpha W463F/beta E190Q/beta Y341W), also gave the same results. The half-maximal concentrations of P(i) that suppressed the MgADP-inhibited form and interfered with MgATP binding were both approximately 20 mm. It is likely that the presence of P(i) at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme-MgADP-P(i) complex, an active intermediate in the catalytic cycle.
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan.
authors.forename: Noriyo
authors.initials: N
authors.lastname: Mitome
authors.forename: Sakurako
authors.initials: S
authors.lastname: Ono
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Suzuki
authors.forename: Katsuya
authors.initials: K
authors.lastname: Shimabukuro
authors.forename: Eiro
authors.initials: E
authors.lastname: Muneyuki
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D010710:Phosphates / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases