title: Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase. In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate.
abstract: The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z. (1985) FEBS Lett. 178, 10-14). Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E. coli F1-ATPase. Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supported assembly and TNP-ADP did not. The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007293:Inosine Triphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D046911:Macromolecular Substances; D007293:Inosine Triphosphate; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; C035319:2',3' O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases