Substitution of βGlu201 in the α3β3γ subcomplex of the F1-ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides

In the crystal structure of bovine mitochondrial F-1-ATPase (MF1) (Abrahams, J, P,, Leslie, A. G, W., Lutter, R., and Walker, J, E, (1994) Nature 370, 621-628), the side chain oxygen of beta Thr(163) interacts directly with Mg2+ coordinated to 5'-adenylyl beta,gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of beta Thr(163) is hydrogen-bonded to the carboxylate of beta Glu199. Substitution of beta Glu(201) (equivalent to beta Glu199 in MF1) in the alpha(3)beta(3)gamma subcomplex of the F-1-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 mu M ATP, These substitutions lower K-m3 (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively, Fluorescence quenching of alpha(3)(beta E201C/Y341W)(3)gamma and alpha(3)(beta Y341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of beta Thr(163) and beta Glu(199) in the unliganded catalytic site in the crystal structure of MF1 stabilizes the open conformation of the catalytic site during ATP hydrolysis.