C-Terminal Mutations in the Chloroplast ATP Synthase Subunit Impair ATP Synthesis and Stimulate ATP Hydrolysis
详细信息 查看全文
- 作者：Feng He ; Hardeep S. Samra ; Eric A. Johnson ; Nicholas R. Degner ; Richard E. McCarty ; Mark L. Richter
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 15, 2008
- 年：2008
- 卷：47
- 期：2
- 页码：836 - 844
- 全文大小：112K
- 年卷期：v.47,no.2(January 15, 2008)
- ISSN：1520-4995
文摘
Two highly conserved amino acid residues, an arginine and a glutamine, located near theC-terminal end of the 
subunit, form a "catch" by hydrogen bonding with residues in an anionic loop onone of the three catalytic 
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 criticalrole in the binding change mechanism whereby binding of ATP to one catalytic site releases the catchand induces a partial rotation of the subunit. This role is supported by the observation that mutation ofthe equivalent arginine and glutamine residues in the Escherichia coli F1 subunit drastically reduced allATP-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 residuesin the chloroplast F1 subunit, arginine 304 and glutamine 305, with alanine decreased the level ofproton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATPhydrolysis 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, ATPaseactivity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicatingselective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catchresidues are required for efficient proton coupling and for activation of multisite catalysis when MgATPis the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore,for rotation of the subunit.
subunit, form a "catch" by hydrogen bonding with residues in an anionic loop onone of the three catalytic 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 criticalrole in the binding change mechanism whereby binding of ATP to one catalytic site releases the catchand induces a partial rotation of the subunit. This role is supported by the observation that mutation ofthe equivalent arginine and glutamine residues in the Escherichia coli F1 subunit drastically reduced allATP-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 residuesin the chloroplast F1 subunit, arginine 304 and glutamine 305, with alanine decreased the level ofproton-coupled ATP synthesis by more than 80%. Both the Mg2+-dependent and Ca2+-dependent ATPhydrolysis 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, ATPaseactivity of the double mutant was insensitive to inhibition by the phytotoxic inhibitor tentoxin, indicatingselective loss of catalytic cooperativity in the presence of Mg2+ ions. The results indicate that the catchresidues are required for efficient proton coupling and for activation of multisite catalysis when MgATPis the substrate. The catch is not, however, required for CaATP-driven multisite catalysis or, therefore,for rotation of the subunit.