The α3(βMet222Ser/Tyr345Trp)3γ subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity

The alpha(3)(ss(MS)-S-222/(YW)-W-345)(3)gamma double-mutant subcomplex of the F-1-ATPase from the thermophilic Bacillus PS3 (TF1), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K-m of 31 mu M and a k(cat) of 220 s(-1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K-d1 values of less than 1 nM and bind to the site of intermediate affinity with a common K-d2 value of about 12 nM. The K-d3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 mu M, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(ss(MS)-S-2822/(YW)-W-345)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [H-3] ADP remains bound when the wild-type and doublemutant subcomplexes hydrolyze substoichiometric [H-3] ATP. In contrast, (32)Pi is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-P-32] ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF1 subcomplex hydrolyzes substoichiometric ATP.