TAILIEUCHUNG - Báo cáo Y học: The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F1-ATPase

F1-ATPase is inactivated by entrapment of MgADP in catalytic sites and , usinga mutant a3b3c complex of thermophilic F1-ATPase (aW463F/bY341W) and monitoring nucleotide binding by ¯uorescence quenching of an introduced tryptophan, we found that P i interfered with the binding of MgATP to F1-ATPase, but binding ofMgADPwas interferedwith to a lesser extent. | Eur. J. Biochem. 269 53 60 2002 FEBS 2002 The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F1-ATPase Noriyo Mitome Sakurako Ono Toshiharu Suzuki Katsuya Shimabukuro Eiro Muneyuki and Masasuke Yoshida Chemical Resources Laboratory Tokyo Institute of Technology Japan Fi-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or Pi. Here using a mutant a3p3c complex of thermophilic F1-ATPase aW463F bY341W and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan we found that Pi interfered with the binding of MgATP to F1-ATPase but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F1-ATPase during the experiments did not obscure the interpretation because another mutant which was able to bind nucleotide but not hydrolyse ATP aW463F bE190Q bY341W also gave the same results. The half-maximal concentrations of Pi that suppressed the MgADP-inhibited form and interfered with MgATP binding were both w 20 mM. It is likely that the presence of Pi at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme MgADP Pi complex an active intermediate in the catalytic cycle. Keywords competition FoF1-ATP synthase MgADP inhibition nucleotide binding tryptophan fluorescence. FoF1-ATP synthase synthesizes ATP from ADP and inorganic phosphate Pi by using the energy of proton flow driven by a transmembrane electrochemical proton gradient 1 3 . The enzyme consists of a cytoplasmic domain referred to as F1-ATPase or F1 which carries catalytic sites for the synthesis and hydrolysis of ATP and a membrane integral domain Fo which conducts protons across the membrane. F1 has a subunit composition a3p3yỗe and can be reversibly separated from Fo. There are six nucleotide-binding sites in F1-ATPase three of which are catalytic sites located on the b subunits and three other noncatalytic sites located on the a subunits. In the

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