Structural Asymmetry of F1-ATPase Caused by the γ Subunit Generates a High Affinity Nucleotide Binding Site

Abstract The α3β3γ and α3β3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the α3β3 complex. The results of equilibrium dialysis show that the α3β3γ complex has a high affinity nucleotide binding site and several low affinity sites, whereas the α3β3 complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the β subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the α3β3γ complex is accelerated by the chase addition of excess ATP, whereas that by the α3β3 complex is not. We further examined the complexes containing mutant β subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant β subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K., Yutani, K., and Yoshida, M.(1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the β subunit in the mutant α3β3γ complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant α3β3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the γ subunit is responsible for the generation of a high affinity nucleotide binding site on the β subunit in F1-ATPase where cooperative catalysis can proceed.