Adenosinetriphosphate (ATP) synthase (FoF
1) is a major energy supplying enzyme of cells utilizing the proton motive force. It consists of a catalytic portion called F
1 and a proton channel portion called Fo. In order to elucidate the chemical reaction of FoF
1, thermophilic FoF
1 (TFoF
1) was used, because it is stable and could eb reconstituted without Mg-ATP. In contrast to the previous hypotheses on the ATP synthesis, direct measurement of H
+ current through TFoF
1 incorporated into a planar lipid bilayer, 3H
+/ATP stoichiometry was obtained. The primary structure of TFoF
1 was established by sequencing its operon deoxyribonucleic acid and subunit peptides. The stereochemistry of the reaction using [
16O,
17O,
15O,
35S] thiophosphate supported the a pathway for associative nucleophilic displacement on a phosphoric ester without pseudorotation. The diastereoisomeric preference of Cd-ATPγS revealed that the true substrate of TFoF
1 is Δ, β, γ, bidentate Mg-ATP, like adenylate kinase. 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. Mitchell's chemiosmotic theory was refined by these results.
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