Catalytic activities of alpha3beta3gamma 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 F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha3beta3gamma 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 alpha3beta3gamma complexes, one containing beta(wild-type) and the other Glu.Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha3beta3gamma 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).
Amano
T
T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan.
Hisabori
T
T
Muneyuki
E
E
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry
C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases