Mutation of the α subunit of the Escherichia coli F1-ATPase to convert Arg-376 to a Cys (αR376C) lowers multisite ATPase activity 400-1,000-fold while affecting unisite catalysis only around 6-fold, suggesting that the mutation is in a region important for transmission of conformational changes between catalytic sites (Soga, S., Noumi, T., Takeyama, M., Maeda, M., and Futai, M. (1989) Arch. Biochem. Biophys. 268, 643-648; this study). To learn more of the structural features of the segment of the α subunit around Arg- 376, mutant enzyme with a Cys at this position was modified with several maleimides. N-[14C]Ethylmaleimide reacted rapidly with this Cys in one of the three α subunits/F1 (2,500 M-1 s-1); more slowly with a second α subunit (390 M-1 s-1); and the same Cys in the third copy of the α subunit was completely unreactive to the reagent, indicating asymmetry of α subunits in the ECF1 complex. The photoactivatable cross-linker N-(4-azido- 2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide, when reacted via its maleimide to αCys-376 of the mutant, covalently linked α to β subunits upon photolysis, indicating that Cys-376 of α is close to an interface between the α and β subunits. The EDTA-induced exchangeable noncatalytic site could be filled by TNP-ATP in both wild type and αR376C mutant ECF1. Occupancy of this site in the αR376C mutant altered the rate of reaction of the second-fastest reacting Cys-376 from 390 M-1 s-1 to below 130 M-1 s-1, suggesting that the two sites are on the same α subunit. TNP-ATP in the EDTA-induced exchangeable noncatalytic site was quenched by reacting Cys- 376 with 4-maleimido-(2,2,6,6-tetramethylpiperidine-N(oxyl), indicating that the region around Cys-376, which is involved in transmission of conformational changes between α and β, and noncatalytic sites are maximally 10-12 Å from each other.
Turina P., Aggeler R., Lee R.S.F., Senior A.E., Capaldi R.A. (1993). The cysteine introduced into the α subunit of the Escherichia coli F1- ATPase by the mutation αR376C is near the α-β subunit interface and close to a noncatalytic nucleotide binding site. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 268(10), 6978-6984.
The cysteine introduced into the α subunit of the Escherichia coli F1- ATPase by the mutation αR376C is near the α-β subunit interface and close to a noncatalytic nucleotide binding site
Turina P.;
1993
Abstract
Mutation of the α subunit of the Escherichia coli F1-ATPase to convert Arg-376 to a Cys (αR376C) lowers multisite ATPase activity 400-1,000-fold while affecting unisite catalysis only around 6-fold, suggesting that the mutation is in a region important for transmission of conformational changes between catalytic sites (Soga, S., Noumi, T., Takeyama, M., Maeda, M., and Futai, M. (1989) Arch. Biochem. Biophys. 268, 643-648; this study). To learn more of the structural features of the segment of the α subunit around Arg- 376, mutant enzyme with a Cys at this position was modified with several maleimides. N-[14C]Ethylmaleimide reacted rapidly with this Cys in one of the three α subunits/F1 (2,500 M-1 s-1); more slowly with a second α subunit (390 M-1 s-1); and the same Cys in the third copy of the α subunit was completely unreactive to the reagent, indicating asymmetry of α subunits in the ECF1 complex. The photoactivatable cross-linker N-(4-azido- 2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide, when reacted via its maleimide to αCys-376 of the mutant, covalently linked α to β subunits upon photolysis, indicating that Cys-376 of α is close to an interface between the α and β subunits. The EDTA-induced exchangeable noncatalytic site could be filled by TNP-ATP in both wild type and αR376C mutant ECF1. Occupancy of this site in the αR376C mutant altered the rate of reaction of the second-fastest reacting Cys-376 from 390 M-1 s-1 to below 130 M-1 s-1, suggesting that the two sites are on the same α subunit. TNP-ATP in the EDTA-induced exchangeable noncatalytic site was quenched by reacting Cys- 376 with 4-maleimido-(2,2,6,6-tetramethylpiperidine-N(oxyl), indicating that the region around Cys-376, which is involved in transmission of conformational changes between α and β, and noncatalytic sites are maximally 10-12 Å from each other.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
