|
He F, Samra HS, Johnson EA, Degner NR, McCarty RE, Richter ML.
Biochemistry. 2008 Jan 15;47(2):836-44.
C-Terminal mutations in the chloroplast ATP synthase gamma subunit impair
ATP synthesis and stimulate ATP hydrolysis.
Department of Molecular Biosciences, The University of Kansas, Lawrence,
Kansas 66045, USA.
Two highly conserved amino acid residues, an arginine and a glutamine,
located near the C-terminal end of the gamma subunit, form a "catch" by hydrogen
bonding with residues in an anionic loop on one of the three catalytic beta
subunits of the bovine mitochondrial F1-ATPase [Abrahams, J. P., Leslie, A. G.,
Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628]. The catch is
considered to play a critical role in the binding change mechanism whereby
binding of ATP to one catalytic site releases the catch and induces a partial
rotation of the gamma subunit. This role is supported by the observation that
mutation of the equivalent arginine and glutamine residues in the Escherichia
coli F1 gamma subunit drastically reduced all ATP-dependent catalytic activities
of the enzyme [Greene, M. D., and Frasch, W. D. (2003) J. Biol. Chem. 278,
5194-5198]. In this study, we show that simultaneous substitution of the
equivalent residues in the chloroplast F1 gamma subunit, arginine 304 and
glutamine 305, with alanine decreased the level of proton-coupled ATP synthesis
by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATP hydrolysis
activities increased by more than 3-fold as a result of these mutations;
however, the sulfite-stimulated activity decreased by more than 60%. The
Mg2+-dependent, but not the Ca2+-dependent, ATPase activity of the double mutant
was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicating
selective loss of catalytic cooperativity in the presence of Mg2+ ions. The
results indicate that the catch residues are required for efficient proton
coupling and for activation of multisite catalysis when MgATP is the substrate.
The catch is not, however, required for CaATP-driven multisite catalysis or,
therefore, for rotation of the gamma subunit.
PMID: 18092810
|
