Substitution of βGLu201 in the α3β3γ subcomplex of the F1- ATPase from the thermophilic Bacillus PS3 increases the affinity of catalytic sites for nucleotides

Abstract

In the crystal structure of bovine mitochondrial F1-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 βThr163 interacts directly with Mg2+ coordinated to 5'-adenylyl β,γ-imidodiphosphate or ADP bound to catalytic sites of β subunits present in closed conformations. In the unliganded β subunit present in an open conformation, the hydroxyl of βThr163 is hydrogen-bonded to the carboxylate of βGlu199. Substitution of βGlu201 (equivalent to βGlu199 in MF1) in the α3β3γ subcomplex of the F1-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 μM 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 α3(βE201C/Y341W)3γ and α3(βY341W)3γ 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 thai the hydrogen bond observed between the side chains of βThr163 and βGlu199 in the unliganded catalytic site in the crystal structure of MF1 stabilizes the open conformation of the catalytic site during ATP hydrolysis.