ATP hydrolyses by the wild-type a&y and mutant (aD261N)3P3y subcomplexes of the FI-ATPase from the thermophilic Bacillus PS3 have been compared. The wild-type complex hydrolyzes 50 p M 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 p M 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 p M 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 p M 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.
The F,Fl-ATP synthases couple ATP synthesis and hydrolysis to proton electrochemical gradients in energytransducing membranes (Senior, 1990;Pedersen & Amzel, 1993). F, is an integral membrane protein complex that mediates proton conduction, whereas F1 is a peripheral membrane protein complex which bears the catalytic sites. When removed from the membrane in soluble form, F1 is an ATPase. The F1-ATPases are comprised of five different subunits in a stoichiometry of a&y& and have molecular weights of about 380000. They contain six nucleotide binding sites, three of which are catalytic (Cross, 1992). The other three, for want of a defined physiological function, are