Location and properties of pyrophosphate-binding sites in Escherichia coli F1-ATPase.
Binding of pyrophosphate (PPi) to the three catalytic ("C") and three noncatalytic ("NC") nucleotide sites of Escherichia coli F1-ATPase was determined by fluorescence spectroscopy using mutant enzymes with tryptophan inserted specifically in either C sites (beta Y331W) or NC sites (alpha R365W). Fluorescence of the tryptophan is quenched on binding of nucleotide; PPi binding parameters were determined by competition with ATP or adenyl-5'-yl imidodiphosphate. It was found that MgPPi binds to each NC site with Kd = 20 microM. In contrast, even at millimolar concentration, neither MgPPi nor free PPi showed significant binding to C sites. We confirmed that free PPi displaces nucleotide from C sites, but this was shown to be due to complexation of Mg2+ ions rather than to occupancy of the sites. MgPPi bound at NC sites was found not to affect ATP hydrolysis rates. From the data we propose a two-phase model for nucleotide binding at NC sites. In phase one, NC sites recognize the pyrophosphate "end" of the nucleotide, which binds initially with Kd similar to MgPPi; in phase two, a slow conformational change occurs which tightly sequesters adenine nucleotide. Phase two does not occur with guanine nucleotide. This model explains the preference of NC sites for adenine nucleotides. Pi (5 mM) did not bind to either C or NC sites.
Weber
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642, USA.
Senior
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AE
D000227:Adenine Nucleotides / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D011756:Diphosphates / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008024:Ligands; D017616:Magnesium Compounds; D008956:Models, Chemical; D016296:Mutagenesis; D010710:Phosphates / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
D000227:Adenine Nucleotides; D011756:Diphosphates; D008024:Ligands; D017616:Magnesium Compounds; D010710:Phosphates; D000266:Adenylyl Imidodiphosphate; D000249:Adenosine Monophosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; C034732:magnesium pyrophosphate; D006180:Proton-Translocating ATPases