F1-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 F1-ATPase catalytic sites, were investigated. The mutations betaK155Q, betaD242N, and betaE181Q were each combined with the betaY331W 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.