Catalytic activities of alpha(3)beta(3)gamma complexes of F-1-ATPase with 1, 2, or 3 incompetent catalytic sites

In order to know how many functional catalytic sites are necessary for ATPase activity of F-1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha(3) beta(3) gamma complex with 1, 2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu . Tag) were linked to the C terminus of the catalytically incompetent beta(E190Q) subunit. The Glu Tag itself did not affect ATPase activity of the complexes. Two kinds of alpha(3) beta(3) gamma complexes, one containing beta(wild-type) and the other Glu Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha(3) beta(3) gamma complexes were reconstituted. Each of the complexes containing a different number of Glu . Tag-linked beta(E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M. (1989) J. Biochem. (Tokyo) 106, 730-734).