The role of β-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase

beta-Arg-182 in Escherchia coli F-1-ATPase (beta-Arg-189 in bovine mitochondrial F-l) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, beta R182Q and beta R182K. Oxidative phosphorylation and steady-state ATPase activity of purified F-l were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F-l from beta R182Q/beta Y331W and beta R182K/beta Y331W double mutants. It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155. A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis.