遺伝子組換えによるアデノシン三リン酸合成酵素の化学反応の解析

概要

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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 [^<16>O, ^<17>O, ^<15>O, ^<35>S] 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.
公益社団法人日本薬学会の論文
1987-11-25