title: Binding of phytopolyphenol piceatannol disrupts β/γ subunit interactions and rate-limiting step of steady-state rotational catalysis in Escherichia coli F1-ATPase.
abstract: In observations of single molecule behavior under V(max) conditions with minimal load, the F(1) sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (∼0.2 ms) and 120° rotation steps (∼0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F(1) also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met(23) with Lys, indicating that the inhibitor and the β/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, and Futai Special Laboratory, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Sekiya
authors.forename: Robert K
authors.initials: RK
authors.lastname: Nakamoto
authors.forename: Mayumi
authors.initials: M
authors.lastname: Nakanishi-Matsui
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Futai
mesh_terms: D000595:Amino Acid Sequence; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D059808:Polyphenols / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011794:Quercetin / Q000378:metabolism / Q000494:pharmacology; D013267:Stilbenes / Q000378:metabolism* / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000975:Antioxidants; D029968:Escherichia coli Proteins; D059808:Polyphenols; D021122:Protein Subunits; D013267:Stilbenes; C041525:3,3',4,5'-tetrahydroxystilbene; D011794:Quercetin; D006180:Proton-Translocating ATPases