The trapping of different conformations of the Escherichia coli F1 ATPase by disulfide bond formation. Effect on nucleotide binding affinities of the catalytic sites.

The Journal of Biological Chemistry
G Grüber, R A Capaldi

Abstract

Two mutants of the Escherichia coli F1 ATPase, betaY331W:E381C/epsilonS108C and alphaS411C/betaY331W/epsilonS108C, have been used to relate nucleotide binding in catalytic sites with different interactions of the stalk-forming subunits gamma and epsilon at the alpha3beta3 subunit domain. Essentially full yield cross-linking between beta + gamma and beta + epsilon, or between alpha + gamma and alpha + epsilon, was obtained in these mutants by Cu2+-induced disulfide bond formation, thereby trapping the enzyme in states with the small subunits interacting either with beta or alpha subunits. The presence of the Trp for beta Tyr-331 in both mutants allowed direct measurement of nucleotide occupancy of catalytic sites. Before cross-linking, Mg2+ATP could be bound in all three catalytic sites in both mutants with a Kd of around 0.1 microM for the highest affinity site and Kd values of approximately 2 microM and 30-40 microM for the second and third sites, respectively. In the absence of Mg2+, ATP also bound in all three catalytic sites but with a single low affinity (above 100 microM) in both mutants. Cu2+-induced cross-linking of ECF1 from the mutant betaY331W:E381C/epsilonS108C had very little effect on nucleotide binding. The bindi...Continue Reading

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Citations

Jul 20, 1999·Annual Review of Biophysics and Biomolecular Structure·R K NakamotoM K al-Shawi
Nov 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·A C HausrathR A Capaldi
Dec 13, 2005·FEBS Letters·Yasuyuki Kato-Yamada
Sep 20, 2006·Biochemical and Biophysical Research Communications·Satoshi IizukaYasuyuki Kato-Yamada
Feb 20, 1999·The Journal of Biological Chemistry·S P TsunodaH Noji
Oct 18, 2000·FEBS Letters·J WeberA E Senior
Jul 3, 2003·The Journal of Biological Chemistry·Yasuyuki Kato-Yamada, Masasuke Yoshida
Jun 20, 1998·The Journal of Biological Chemistry·J J García, R A Capaldi
Dec 6, 1997·Biochemical and Biophysical Research Communications·Y Kagawa, T Hamamoto
May 9, 2002·Biochimica Et Biophysica Acta·Alan E SeniorJoachim Weber
Jun 6, 2000·Biochimica Et Biophysica Acta·R K NakamotoM K Al-Shawi
Jun 6, 2000·Biochimica Et Biophysica Acta·H Ren, W S Allison
Apr 1, 2007·EcoSal Plus·Steven B Vik

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