The alpha 3 beta 3 gamma complex of the F1-ATPase from thermophilic Bacillus PS3 containing the alpha D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites.
ATP hydrolyses by the wild-type alpha 3 beta 3 gamma and mutant (alpha D261N)3 beta 3 gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 microM ATP in three kinetic phases: a burst decelerates to an intermediate phase, which then gradually accelerates to a final rate. In contrast, the mutant complex hydrolyzes 50 microM or 2 mM ATP in two kinetic phases. The mutation abolishes acceleration from the intermediate phase to a faster final rate. Both the wild-type and mutant complexes hydrolyze ATP with a lag after loading a catalytic site with MgADP. The rate of the MgADP-loaded wild-type complex rapidly accelerates and approaches that observed for the wild-type apo-complex. The MgADP-loaded mutant complex hydrolyzes ATP with a more pronounced lag, and the gradually accelerating rate approaches the slow, final rate observed with the mutant apo-complex. Lauryl dimethylamide oxide (LDAO) stimulates hydrolysis of 2 mM ATP catalyzed by wild-type and mutant complexes 4- and 7.5-fold, respectively. The rate of release of [3H]ADP from the Mg[3H]ADP-loaded mutant complex during hydrolysis of 40 microM ATP is slower than observed with the wild-type complex. LDAO increases the rate of release of [3H]ADP from the preloaded wild-type and mutant complexes during hydrolysis of 40 microM ATP. Again, release is slower with the mutant complex. When the wild-type and mutant complexes are irradiated in the presence of 2-N3-[3H]ADP plus Mg2+ or 2-N3-[3H]ATP plus Mg2+ and azide, the same extent of labeling of noncatalytic sites is observed. Whereas ADP and ATP protect noncatalytic sites of the wild-type and mutant complexes about equally from labeling by 2-N3-[3H]ADP or 2-N3-[3H[ATP, respectively, AMP-PNP provides little protection of noncatalytic sites of the mutant complex. The results suggest that the substitution does not prevent binding of ADP or ATP to noncatalytic sites, but rather that it affects cross-talk between liganded noncatalytic sites and catalytic sites which is necessary to promote dissociation of inhibitory MgADP.
Jault
J M
JM
Department of Chemistry and Biochemistry 0601, University of California at San Diego, La Jolla 92093-0601, USA.
Matsui
T
T
Jault
F M
FM
Kaibara
C
C
Muneyuki
E
E
Yoshida
M
M
Kagawa
Y
Y
Allison
W S
WS
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D004123:Dimethylamines / Q000494:pharmacology; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines / Q000494:pharmacology; D013329:Structure-Activity Relationship
D004123:Dimethylamines; D012235:Rhodamines; C026188:rhodamine 6G; D000266:Adenylyl Imidodiphosphate; C014518:dodecyldimethylamine oxide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases