C. Kaibara et al., STRUCTURAL ASYMMETRY OF F1-ATPASE CAUSED BY THE GAMMA-SUBUNIT GENERATES A HIGH-AFFINITY NUCLEOTIDE-BINDING SITE, The Journal of biological chemistry, 271(5), 1996, pp. 2433-2438
The alpha(3) beta(3) gamma and alpha(3)beta>(3) complexes of F-1-ATPas
e from a thermophilic Bacillus PS3 were compared in terms of interacti
on with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) th
at differed from ATP and ADP and did not destabilize the alpha(3) beta
(3) complex. The results of equilibrium dialysis show that the alpha(3
) beta(3) gamma complex has a high affinity nucleotide binding site an
d several low affinity sites, whereas the alpha(3) beta(3) complex has
only low affinity sites. This is also supported from analysis of spec
tral change induced by TNP-ADP, which in addition indicates that this
high affinity site is located on the beta subunit. Single-site hydroly
sis of substoichiometric amounts of TNP-ATP by the alpha(3) beta(3) ga
mma complex is accelerated by the chase addition of excess ATP, wherea
s that by the alpha(3) beta(3) complex is not. We further examined the
complexes containing mutant beta subunits (Y341L, Y341A, and Y341C).
Surprisingly, in spite of very weak affinity of the isolated mutant be
ta 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 beta subunit in the mutant alpha(3) beta(3) gamma
complexes where single-site TNP-ATP hydrolysis can occur. ATP concent
rations required for the chase acceleration of the mutant complexes ar
e higher than that of the wild-type complex. The mutant alpha(3) beta(
3) complexes, on the contrary, catalyze single-site hydrolysis of TNP-
ATP rather slowly, and there is no chase acceleration. Thus, the gamma
subunit is responsible for the generation of a high affinity nucleoti
de binding site on the beta subunit in F-1-ATPase where cooperative ca
talysis can proceed.