Abstract
ATP synthase (F0F1) is a major energy supplying enzyme of cells utilizing the proton motive force across the biomembrane. It consists of a catalytic portion called F1 and a proton channel portion called F0. For elucidation of the chemical reaction of F0F1, thermophilic F0F1 (TF0F1) was used, because it is stable and could be reconstituted without Mg-ATP. A new method for incorporating F0F1 in a planar lipid bilayer was developed by using a lipid monolayer technique and liposome fusion. Then the electrogenic proton translocation of F0F1 was directly measured. In contrast to the previous hypotheses on the ATP synthesis, direct measurement of II+ current through TF0F1 incorporated into a planar lipid bilayer, 3H+/ATP stoichiometry was obtained. The primary structure of TF0F1 was established by sequencing its operon DNA and subunit peptides. The stereochemistry of the reaction using [16O, 17O, 18O, 35O] thiophosphate supported the a pathway for associative nucleophilic displacement on a phosphoric ester. The site directed mutagenesis of the residues of F1 homologous to Mg-ATP binding site of adenylate kinase revealed their essential role in the reaction.
