ATP synthase (F
0F
1) is a major energy supplying enzyme of cells utilizing the proton motive force across the biomembrane. It consists of a catalytic portion called F
1 and a proton channel portion called F
0. For elucidation of the chemical reaction of F
0F
1, thermophilic F
0F
1 (TF
0F
1) was used, because it is stable and could be reconstituted without Mg-ATP. A new method for incorporating F
0F
1 in a planar lipid bilayer was developed by using a lipid monolayer technique and liposome fusion. Then the electrogenic proton translocation of F
0F
1 was directly measured. In contrast to the previous hypotheses on the ATP synthesis, direct measurement of II+ current through TF
0F
1 incorporated into a planar lipid bilayer, 3H+/ATP stoichiometry was obtained. The primary structure of TF
0F
1 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 F
1 homologous to Mg-ATP binding site of adenylate kinase revealed their essential role in the reaction.
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