TRYPTOPHAN SUBSTITUTIONS SURROUNDING THE NUCLEOTIDE IN CATALYTIC SITES OF F-1-ATPASE

Novel tryptophan substitutions, surrounding the nucleotide bound in ca talytic sites, were introduced into Escherichia codi F-1-ATPase. The m utant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp -410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-1 53 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide bindin g pattern with MgADP and MgAMPPNP, with one site of higher and two sit es of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinit y, and the region of the catalytic site sensed by these Trp substituti ons did not change conformation with different nucleotides. In contras t, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-st ate, V-max MgATP hydrolysis.