Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated β-subunit of Escherichia coli F₁-ATPase. In vitro assembly of F₁-subunits requires occupancy of the nucleotide-binding site on β-subunit by nucleoside triphosphate

The stoichiometry of nucleotide binding to the isolated α- and β-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 α-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol β-subunit, consistent with previous equilibrium dialysis studies which showed isolated β-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 ~0.1 mol of ATP or ADP per mol of β-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the α- and β-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is α₃β₃γδε, 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 α-, β-, and γ-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 μM, 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 β-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the α-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.