CATALYTIC ACTIVITIES OF ALPHA(3)BETA(3)GAMMA COMPLEXES OF F1-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 develope d. 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) be ta(3) gamma complexes, one containing beta(wild-type) and the other Gl u Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, an d alpha(3) beta(3) gamma complexes were reconstituted. Each of the com plexes containing a different number of Glu . Tag-linked beta(E190Q) w as separated by anion-exchange chromatography and analyzed. The result s were as follows. 1) Normal steady state ATPase activity requires thr ee intact catalytic sites. 2) Chase-acceleration, a catalytic cooperat ivity, requires at least two intact catalytic sites. 3) Single-site ca talysis can be mediated by a single intact catalytic site alone. Rescr ambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons f or previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., H isabori, T., Date, T., and Yoshida, M. (1989) J. Biochem. (Tokyo) 106, 730-734).