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Investigating the Rotational Catalytic Mechanism of the Escherichia Coli F-ATPase

Baylis Scanlon, Joanne Amanda
Thesis/Dissertation; Online
Baylis Scanlon, Joanne Amanda
Shao, Zhifeng
Guilford, William
Tamm, Lukas
Egelman, Edward
Nakamoto, Robert
The F O F 1 is a multi-subunit enzyme that functions as a mechanical motor using rotation to efficiently couple the chemical energy from ATP synthesis/hydrolysis to ion translocation across the membrane. Steady state ATP hydrolysis in the F1-ATPase involves rotation of the central  subunit relative to the catalytic sites in the 33 pseudo hexamer. In order to understand the role of γ subunit rotation in the catalytic mechanism, the pre-steady state kinetics of Mg·ATP hydrolysis upon rapid filling of all three catalytic sites was determined in the F 1 -ATPase. The experimentally accessible partial reactions leading up to the rate limiting step and continuing through to the steady state mode were obtained for the first time. Analysis of the burst kinetics of Mg·ATP hydrolysis indicated that the rate limiting step follows hydrolysis and precedes the release of products Pi and ADP. The burst kinetics and steady state hydrolysis for a range of Mg·ATP concentrations provided adequate constraints for a unique minimal kinetic model which can fit all the data and satisfied extensive sensitivity tests. Consistent with the single molecule analysis of Yasuda et al. [Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., and Itoh, H. (2001) Nature 410, 898-904], we propose that the rate-limiting step involves a partial rotation of the γ subunit; hence this step was named k γ . Moreover, our model suggests that reversible hydrolysis/synthesis can only occur in the active site of the β TP conformer [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. To directly test the model two single cysteine mutants, βD380C and βE381C, were used, which can reversibly inhibit rotation upon formation of a cross-link with the ii conserved γCys87. In the pre-steady state, the γ-β cross-linked enzyme at high Mg·ATP conditions retained the burst of hydrolysis but was not able to release Pi. These data show that the rate-limiting rotation step, k γ , occurs after hydrolysis and before Pi release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the βGlu381 residue for proper formation of the rate-limiting transition state involving γ subunit rotation. Note: Abstract extracted from PDF text
University of Virginia, Department of Biophysics, PHD, 2008
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