Modulation of nucleotide specificity of thermophilic FoF 1-ATP synthase by ε-subunit

Abstract

The C-terminal two α-helices of the ε-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm ("up-state") and a retracted hairpin ("down-state"). As ATP becomes poor, ε changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ε in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the upstate ε was achieved by inclusion of hexokinase in the assay and TFoF1 with the down-state ε was represented by εΔc-TFoF1, in which ε lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TFoF1 with the down-state ε synthesizes GTP at the same rate of ATP, whereas TFoF1 with the up-state ε synthesizes GTP at a halfrate. Though rates are slow, TF oF1 with the down-state ε even catalyzes UTP and CTP synthesis. Authentic TFoF1 from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the downstate. NTP hydrolysis and NTP-driven proton pumping activity of εΔc-TFoF1 suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ε-subunit modulates substrate specificity of TF oF1. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.