title: Studies of nucleotide binding to the catalytic sites of Escherichia coli betaY331W-F1-ATPase using fluorescence quenching.
abstract: Most studies of nucleotide binding to catalytic sites of Escherichia coli betaY331W-F(1)-ATPase by the quenching of the betaY331W fluorescence have been conducted in the presence of approximately 20 mM sulfate. We find that, in the absence of sulfate, the nucleotide concentration dependence of fluorescence quenching induced by ADP, ATP, and MgADP is biphasic, revealing two classes of binding sites, each contributing about equally to the overall extent of quenching. For the high-affinity catalytic site, the K(d) values for MgADP, ADP, and ATP equal 10, 43, and 185 nM, respectively. For the second class of sites, the K(d) values for these ligands are approximately 1,000x larger at 8.1, 37, and 200 microM, respectively. The presence of sulfate or phosphate during assay results in a marked increase in the apparent K(d) values for the high-affinity catalytic site. The results show, contrary to earlier reports, that Mg(2+) is not required for expression of different affinities for a nucleotide by the three catalytic sites. In addition, they demonstrate that the fluorescence of the introduced tryptophans is nearly completely quenched when only two sites bind nucleotide. Binding of ADP to the third site with a K(d) near mM gives little fluorescence change. Many previous results of fluorescence quenching of introduced tryptophans appear to require reinterpretation. Our findings support a bi-site catalytic mechanism for F(1)-ATPase.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Bulygin
authors.forename: Yakov M
authors.initials: YM
authors.lastname: Milgrom
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D008024:Ligands; D009711:Nucleotides; D010710:Phosphates; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium