F-1-ATPase, roles of three catalytic site residues

Three critical residues, beta-Lys-155, beta-Asp-242, and beta-GLu-181, situated close to the gamma-phosphate of MgATP in F-1-ATPase catalytic sites, were investigated. The mutations beta K155Q, beta D242N, and beta E181Q were each combined with the beta Y331W mutation; the fluorescence signal of beta-Trp-331 was used to determine MgATP, MgADP, ATP, and ADP binding parameters for the three catalytic sites of the enzyme. The quantitative contribution of side chains to binding energy at all three catalytic sites was calculated. The following conclusions were made. The major functional interaction of beta-Lys-155 is with the gamma-phosphate of MgATP and is of primary importance at site 1 (the site of highest affinity) and site 2. Release of MgATP during oxidative phosphorylation requires conformational re-positioning of this residue. The major functional interaction of beta-Asp-242 is with the magnesium of the magnesium nucleotide at site 1; it has little or no influence at site 2 or 3. In steady-state turnover, the MgATP hydrolysis reaction occurs at site 1. beta-Glu-181 contributes little to nucleotide binding; its major catalytic effect derives apparently from a role in reaction chemistry per se. This work also emphasizes that nucleotide binding cooperativity shown by the three catalytic sites toward MgATP and MgADP is absolutely dependent on the presence of magnesium.