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• W2013906099 abstract "The F0F1-ATP synthase couples the functions of H+ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located γ, ϵ, and c subunits. To understand the γ subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F1-ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, βD380C and βE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved γCys-87. 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, kγ, occurs after hydrolysis and before Pi release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the βGlu-381 residue for proper formation of the rate-limiting transition state involving γ subunit rotation. The F0F1-ATP synthase couples the functions of H+ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located γ, ϵ, and c subunits. To understand the γ subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F1-ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, βD380C and βE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved γCys-87. 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, kγ, occurs after hydrolysis and before Pi release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the βGlu-381 residue for proper formation of the rate-limiting transition state involving γ subunit rotation. The F0F1-ATP synthase, located in energy-transducing membranes, utilizes the electrochemical gradient of H+ or Na+ to synthesize ATP from ADP and Pi. Under anaerobic conditions, the bacterial enzyme can function as an ATPase, coupled to pump protons across the membrane to generate a ΔμH+. Proton transport is mediated by the membrane-embedded F0 sector (ab2c10) and is separated by a distance greater than 120 Å from the three catalytic sites where ATP hydrolysis/synthesis occurs on the membrane extrinsic F1 segment (α3β3γδϵ) (for reviews see Refs. 1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1571) Google Scholar, 2Nakamoto R.K. Ketchum C.J. Kuo P.H. Peskova Y.B. Al-Shawi M.K. Biochim. Biophys. Acta. 2000; 1458: 289-299Crossref PubMed Scopus (36) Google Scholar, 3Boyer P.D. FEBS Lett. 2002; 512: 29-32Crossref PubMed Scopus (68) Google Scholar, 4Weber J. Senior A.E. FEBS Lett. 2003; 545: 61-70Crossref PubMed Scopus (236) Google Scholar, 5Junge W. Panke O. Cherepanov D.A. Gumbiowski K. Muller M. Engelbrecht S. FEBS Lett. 2001; 504: 152-160Crossref PubMed Scopus (107) Google Scholar). This distance indicates a complicated mechanism for effectual, efficient transmission between the two disparate functions. It is now well established that the enzyme functions as a molecular motor (6Noji H. Yasuda R. Yoshida M. Kinosita K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1944) Google Scholar, 7Tanabe M. Nishio K. Iko Y. Sambongi Y. Iwamoto-Kihara A. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 15269-15274Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Nishio K. Iwamoto-Kihara A. Yamamoto A. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13448-13452Crossref PubMed Scopus (88) Google Scholar), transmitting energy between F1 and F0 through rotation of the central stalk subunits, γ, ϵ, and the ring of c subunits (for reviews see Refs. 9Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell Biol. 2001; 2: 669-677Crossref PubMed Scopus (700) Google Scholar, 10Nakamoto R.K. Baylis Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (127) Google Scholar).The F1 complex can be reversibly stripped from the membrane and functions as a soluble ATPase. In describing the first high resolution x-ray structure, Abrahams et al. (11Abrahams J.P. Buchanan S.K. van Raaij M.J. Fearnley I.M. Leslie A.G.W. Walker J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9420-9424Crossref PubMed Scopus (137) Google Scholar) named each catalytic β subunit for the nucleotide bound at that site as follows: βTP for the ATP analog AMPPNP, 2The abbreviations used are: AMPPNPadenosine 5′-(β,γ-imido)triphosphateDTNB5,5′-dithiobis(2-nitrobenzoic acid)DTTdl-dithiothreitolMDCC-PBP7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled phosphate-binding proteinPDRMphosphodeoxyri-bomutaseTCEPtris(2-carboxyethyl)phosphine hydrochlorideTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidMOPS4-morpholinepropanesulfonic acid. 2The abbreviations used are: AMPPNPadenosine 5′-(β,γ-imido)triphosphateDTNB5,5′-dithiobis(2-nitrobenzoic acid)DTTdl-dithiothreitolMDCC-PBP7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled phosphate-binding proteinPDRMphosphodeoxyri-bomutaseTCEPtris(2-carboxyethyl)phosphine hydrochlorideTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidMOPS4-morpholinepropanesulfonic acid. βDP for ADP-bound, and the structurally open βE empty site (Fig. 1). The three sites have vastly differing affinities for Mg·ATP (12Weber J. Wilke-Mounts S. Lee R.S.-F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar), Kd1 = 10-10, Kd2 = 10-6, and Kd3 = 10-4 m-1 exhibiting negative cooperativity in nucleotide binding. At low substoichiometric Mg·ATP concentrations, characteristics of the basic catalytic reaction have been elucidated in unisite catalytic conditions (13Grubmeyer C. Cross R.L. Penefsky H.S. J. Biol. Chem. 1982; 257: 12092-12100Abstract Full Text PDF PubMed Google Scholar, 14Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 878-885Crossref PubMed Scopus (39) Google Scholar). At higher substrate concentrations, the enzyme enters the multisite hydrolysis mode, which is 105-106 times faster than unisite catalysis because of the high positive cooperativity among the sites with respect to promotion of catalysis (15Grubmeyer C. Penefsky H.S. J. Biol. Chem. 1981; 256: 3728-3734Abstract Full Text PDF PubMed Google Scholar, 16Cross R.L. Grubmeyer C. Penefsky H.S. J. Biol. Chem. 1982; 257: 12101-12105Abstract Full Text PDF PubMed Google Scholar, 17Wise J.G. Latchney L.R. Ferguson A.M. Senior A.E. Biochemistry. 1984; 23: 1426-1432Crossref PubMed Scopus (61) Google Scholar). The Kd value for the binding of Mg·ATP to the low affinity catalytic site (12Weber J. Wilke-Mounts S. Lee R.S.-F. Grell E. Senior A.E. J. Biol. Chem. 1993; 268: 20126-20133Abstract Full Text PDF PubMed Google Scholar) matches the Km value for Mg·ATP hydrolysis (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar, 19Weber J. Bowman C. Senior A.E. J. Biol. Chem. 1996; 271: 18711-18718Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and the Km value for rotation in the direct observation of single F1 molecules (20Yasuda R. Noji H. Yoshida M. Kinosita K. Itoh H. Nature. 2001; 410: 898-904Crossref PubMed Scopus (704) Google Scholar). Rotational catalysis requires the concerted, sequential participation of all three catalytic sites as they pass through the three different conformations (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar, 21Ariga T. Muneyuki E. Yoshida M. Nat. Struct. Mol. Biol. 2007; 14: 841-846Crossref PubMed Scopus (82) Google Scholar, 22Ren H. Bandyopadhyay S. Allison W.S. Biochemistry. 2006; 45: 6222-6230Crossref PubMed Scopus (17) Google Scholar). For these reasons, we have argued (23Al-Shawi M.K. Ketchum C.J. Nakamoto R.K. Biochemistry. 1997; 36: 12961-12969Crossref PubMed Scopus (64) Google Scholar) that reversible hydrolysis/synthesis of ATP occurs in the βTP site, and product Pi and ADP are released after the site converts from βDP to βE (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar). Further evidence using fluorescence resonance energy transfer confirmed that the βTP site is the high affinity site (24Mao H.Z. Weber J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 18478-18483Crossref PubMed Scopus (32) Google Scholar).Single molecule experiments have observed that each major 120° step in γ subunit rotation occurs in two substeps as follows: the 80° substep that is dependent on Mg·ATP concentration, followed by a 40° substep that is not affected by Mg·ATP concentration (25Shimabukuro K. Yasuda R. Muneyuki E. Hara K.Y. Kinosita K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736Crossref PubMed Scopus (214) Google Scholar). Attempts to elucidate the role of rotation in the enzymatic mechanism using the single molecule approach include analysis of γ subunit substeps (20Yasuda R. Noji H. Yoshida M. Kinosita K. Itoh H. Nature. 2001; 410: 898-904Crossref PubMed Scopus (704) Google Scholar, 25Shimabukuro K. Yasuda R. Muneyuki E. Hara K.Y. Kinosita K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736Crossref PubMed Scopus (214) Google Scholar) and concurrent visualization of the binding and release of fluorescent nucleotide (26Nishizaka T. Oiwa K. Noji H. Kimura S. Muneyuki E. Yoshida M. Kinosita K. Nat. Struct. Mol. Biol. 2004; 11: 142-148Crossref PubMed Scopus (240) Google Scholar). However, the structural and mechanistic coordination of γ subunit rotation and the kinetics of the elemental chemical hydrolysis reaction steps cannot be delineated by these studies, and the chemomechanical coupling of the enzyme remains not fully understood.Using pre-steady state analysis, we recently determined the partial reactions of the multisite ATPase hydrolytic pathway and that the rate-limiting step occurs after the reversible hydrolysis/synthesis step and just prior to release of Pi (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar). Scheme 1 adequately describes the multisite kinetic pathway (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar):SCHEME 1View Large Image Figure ViewerDownload Hi-res image Download (PPT)We conjectured that the rate-limiting step involves the 40° rotation, and therefore we called it kγ. The pre-steady state kinetics of this step were critical in allowing us to determine its order in the reaction scheme, which provided tremendous insight into the rotational catalytic mechanism. Even though we could directly determine several of the partial reaction rate constants, assignment of kγ to the rotation step in the presteady state was not confirmed.In this study we test our model that the rate-limiting step kγ involves rotation of the γ subunit. To assay the rotation steps, we decided to use a rotor-stator, γ subunit to β subunit, disulfide bond, with the premise that a cross-link would block rotation and therefore stop the partial reactions involving rotation. Such disulfides have been previously observed between the native Cys at position 87 in the Escherichia coli γ subunit and Cys substitutions in the conserved 380βDELSEED386 motif, either at βAsp-380 (27Duncan T.M. Zhou Y. Bulygin V. Hutcheon M.L. Cross R.L. Biochem. Soc. Trans. 1995; 23: 736-741Crossref PubMed Scopus (20) Google Scholar) or βGlu-381 (28Aggeler R. Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 9185-9191Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) (Fig. 1). For example, Garcia and Capaldi (29García J.J. Capaldi R.A. J. Biol. Chem. 1998; 273: 15940-15945Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) reported that the γ-β-cross-linked F1 had nearly normal unisite kinetic behavior demonstrating that unisite catalysis is independent of the γ subunit rotation. We reasoned that cross-linking the rotor to the stator would lock the position, or at least greatly restrict the rotation of the γ subunit, and therefore prevent the kinetic steps that require rotation. We report here that our data do indeed show this to be the case. Preventing the rotation results in a slower pre-steady state burst of ATP hydrolysis and blocks the release of Pi. These data are consistent with the functional assignments of the catalytic sites where βTP is the high affinity site carrying out reversible hydrolysis/synthesis, and βDP is the site from which product Pi and ADP are released after it converts to βE (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar). We also found that mild perturbations of the βDELSEED function by βE381C causes uncoupling of catalysis and rotation by affecting the establishment of the optimal catalytic conformation resulting in a bypass in the reaction pathway. In the alternative pathway, γ subunit rotation becomes disengaged from hydrolysis thus perturbing its ability to pump protons.Evidence presented in this study suggests the 40° rotation is associated with kγ, and the enzyme needs to acquire the conformation after the 80° rotation substep to achieve positive cooperativity for the promotion of catalysis.EXPERIMENTAL PROCEDURESF0F1 Strains and Plasmids—The single cysteine mutations (βD380C and βE381C) were introduced into plasmid pUβSE derived from a modified pUC18 vector containing a portion of uncD (γ-subunit gene) between SacI and Eco47III restriction sites (30Ketchum C.J. Al-Shawi M.K. Nakamoto R.K. Biochem. J. 1998; 330: 707-712Crossref PubMed Scopus (75) Google Scholar). Site-directed mutagenesis was performed with the QuickChange kit from Stratagene (La Jolla, CA), using the following primers: βE381C, 5′-CTGAAAGACATCATCGCCACCCTGGGTATGGATTGCCTTTCTGAAGAAGACAAACTGGTGG-3′, and βD380C, 5′-CGCCATCCTGGGTATGTGTGAACTTTCTGAAGAAGACAAACTGG-3′ (where the underlined letters show the converted β subunit codons GAA→TGC and GAT→TGT, respectively). Both mutations were isolated on the SacI to Eco47III fragment and ligated individually into the high copy number plasmid pBWU13 (31Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar). Colonies were screened for the insert with a PCR approach using Taq Master Mix (Qiagen, Valencia, CA). Introduction of each mutation in the expression plasmid was verified by DNA sequencing. Mutant F0F1 complexes were expressed in the atp operon-deleted strain, DK8 (32Klionsky D.J. Brusilow W.S.A. Simoni R.D. J. Bacteriol. 1984; 160: 1055-1060Crossref PubMed Google Scholar).The amino-terminal polyhistidine-tagged ϵ subunit (His-ϵ was expressed in E. coli strain BL21(DE3)pLysS (33Andrews S.H. Peskova Y.B. Polar M.K. Herlihy V.B. Nakamoto R.K. Biochemistry. 2001; 40: 10664-10670Crossref PubMed Scopus (18) Google Scholar) and purified as described previously (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar). All molecular biology manipulations were done according to manufacturers' instructions or according to procedures described in Sambrook et al. (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Preparation and Characterization of Mutant F0F1 Enzymes—Oxidative phosphorylation-dependent growth of mutant strains was determined on minimal defined media containing 0.2% sodium succinate as a sole carbon source (31Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar, 35Senior A.E. Latchney L.R. Fersuson A. Wise J.G. Arch. Biochem. Biophys. 1984; 228: 49-53Crossref PubMed Scopus (54) Google Scholar).Membrane vesicles containing F0F1 were prepared as described previously (36Futai M. Sternweis P.C. Heppel L.A. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 2725-2729Crossref PubMed Scopus (208) Google Scholar) from cells grown to mid-log phase in a minimal salt medium supplemented with 1.1% glucose, amino acids, thiamine, and 50 μg/ml ampicillin at 37 °C (31Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar). F1 expression levels in the membrane preparations were analyzed using a quantitative immunoblot assay using polyclonal antibodies against E. coli F1 α subunits (kindly provided by Dr. Alan Senior, University of Rochester). Purified F1 was used as a standard as described previously (37Al-Shawi M.K. Ketchum C.J. Nakamoto R.K. J. Biol. Chem. 1997; 272: 2300-2306Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The formation of ATP- or NADH-dependent electrochemical gradients of protons across inside-out inner membrane vesicles was assessed by monitoring acridine orange fluorescence quenching at 530 nm as described previously (31Moriyama Y. Iwamoto A. Hanada H. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 22141-22146Abstract Full Text PDF PubMed Google Scholar, 38Shin K. Nakamoto R.K. Maeda M. Futai M. J. Biol. Chem. 1992; 267: 20835-20839Abstract Full Text PDF PubMed Google Scholar).Preparation of F1 Enzymes—Cells for F1 purification were grown at 37 °C in minimal media supplemented with 1.1% glucose, amino acids, thiamine, and 50 μg/ml ampicillin. F1 was isolated and purified as described (14Al-Shawi M.K. Senior A.E. Biochemistry. 1992; 31: 878-885Crossref PubMed Scopus (39) Google Scholar). The purity and subunit composition of F1 preparations were checked by SDS-PAGE analysis (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206071) Google Scholar) and by comparison of steady state ATP hydrolysis rates with those reported previously (28Aggeler R. Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 9185-9191Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 33Andrews S.H. Peskova Y.B. Polar M.K. Herlihy V.B. Nakamoto R.K. Biochemistry. 2001; 40: 10664-10670Crossref PubMed Scopus (18) Google Scholar, 40Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (458) Google Scholar).Cross-linking of F1 Mutants—The βD380C mutant F1 was cross-linked by reacting with 5,5′-dithiobis-(2-nitrobenzoate) (DTNB) based on conditions described previously (40Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (458) Google Scholar). The enzyme was thawed and stored at room temperature, diluted to ∼2.5 mg/ml, treated with 1 mm tris(2-carboxyethyl) phosphine hydrochloride (TCEP) for 20 min, and then passed over a Sephadex G-50 centrifuge column (41Penefsky H.S. Methods Enzymol. 1979; 56: 527-530Crossref PubMed Scopus (342) Google Scholar) to remove TCEP and nucleotides and to exchange the buffer into Buffer 1 (25 mm TES-KOH, pH 8.0, 1 mm MgSO4, 10% glycerol). Mutant F1 was incubated with 2.5 μm DTNB for 1 h to induce cross-linking. After incubation, the enzyme was again passed over a centrifuge column to remove oxidizing reagent and exchange the buffer into Buffer 2 (25 mm TES-KOH, 0.244 mm MgCl2, 0.2 mm EDTA, pH 7.5, at 25 °C) in preparation for pre-steady state measurements. The βE381C mutant was treated in the same manner as the βD380C mutant, but to induce the γ-β cross-link 50 μm CuCl2 was used for 1 h (29García J.J. Capaldi R.A. J. Biol. Chem. 1998; 273: 15940-15945Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Incubation with DTNB or CuCl2 overnight did not significantly increase cross-linking efficiency nor affect ATPase activity of wild-type F1. Interestingly, neither mutant F1 achieved a maximal cross-linking yield when treated with the oxidizing reagent optimized for the other.Pre-steady State Multisite Hydrolysis of ATP—[γ-32P]ATP hydrolysis was measured in the millisecond time range using a Kintek RQF-3 rapid quench-flow apparatus (Austin, TX) with circulating water temperature control. The uncross-linked F1 was prepared for pre-steady state experiments by removing bound nucleotides and exchanging the enzyme into Buffer 2 as described previously (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar).In the rapid mixing device (1:1 mixing volume ratio), syringe A contained purified F1 (with an additional equimolar concentration of ϵ subunit) (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar) in Buffer 2, and syringe B contained 25 mm TES-KOH, 0.2 mm EDTA, 0.5 mm [γ-32P]ATP, and 0.46 mm MgSO4, pH 7.5, at 25 °C, resulting in final concentrations of 104 and 50 μm for Mg·ATP and free Mg2+, respectively. The samples were processed according to the two-step procedure described previously (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar) to minimize the background due to the high concentration of [γ-32P]ATP in the experiments. The amount of 32Pi generated was determined by Cerenkov counting in 0.2 m Tris base.Unisite Binding and Hydrolysis of ATP—F1 was prepared as described above for the pre-steady state multisite experiments except that the final centrifuge column was equilibrated with unisite buffer (50 mm Tris base, 50 mm MOPS, 4.5 mm K2SO4 and 0.5 mm MgSO4 adjusted to pH 7.5 with H2SO4). Reactions were performed at 23 °C and were initiated by adding 15 μl of [γ-32P]ATP to F1 while rapidly mixing. The final molar mix ratio of ATP to F1 was ∼0.1. The reactions were quenched at varying times with 550 μl of 50 mm Tris-SO4, pH 8.0, 1 mm KH2PO4, 4.5 mm ATP, 2.8 mm MgSO4, 8.2% v/v HClO4. The total 32Pi generated was determined by the acid molybdate precipitation method of Sugino and Miyoshi (42Sugino Y. Miyoshi Y. J. Biol. Chem. 1964; 239: 2360-2364Abstract Full Text PDF PubMed Google Scholar) and Cerenkov counting in 15 ml of 0.2 m Tris base. The rate of [γ-32P]ATP binding was measured by the hexokinase trap method modified slightly from that described previously (29García J.J. Capaldi R.A. J. Biol. Chem. 1998; 273: 15940-15945Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 43Senior A.E. Lee R.S.-F. Al-Shawi M.K. Weber J. Arch. Biochem. Biophys. 1992; 297: 340-344Crossref PubMed Scopus (46) Google Scholar). Briefly, the unisite reactions were started as described above, but at times shown 200 μl of hexokinase solution (unisite buffer with 2 mg/ml hexokinase (purified from Baker's yeast at ∼170 units/mg), 4.4 mg/ml glucose, and an extra 2.5 mm MgSO4) were added, and the reaction was allowed to proceed for 10 s before quenching with 2 n HCl containing 1 mm Pi. Samples were boiled for 7 min to hydrolyze the unreacted [γ-32P]ATP, and the 32Pi was removed by acid molybdate precipitation. The amount of glucose 6-[32P]phosphate formed was determined by measuring the radioactivity of the supernatant by Cerenkov counting in Tris base.Pre-steady State Pi Release—Pi release was followed by the fluorescence intensity change of (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled phosphate-binding protein (MDCC-PBP (44White H.D. Belknap B. Webb M.R. Biochemistry. 1997; 36: 11828-11836Crossref PubMed Scopus (155) Google Scholar, 45Brune M. Hunter J.L. Howell S.A. Martin S.R. Hazlett T.L. Corrie J.E.T. Webb M.R. Biochemistry. 1998; 37: 10370-10380Crossref PubMed Scopus (172) Google Scholar)). Phosphate-binding protein was expressed from E. coli strain ANCC75 (leu purE trp his argG strA phoS64 met thi) harboring plasmid pSN5182/7 (kindly provided by Dr. Martin Webb, MRC, Mill Hill, London, UK) and prepared as described previously (45Brune M. Hunter J.L. Howell S.A. Martin S.R. Hazlett T.L. Corrie J.E.T. Webb M.R. Biochemistry. 1998; 37: 10370-10380Crossref PubMed Scopus (172) Google Scholar). Unreacted label was separated from MDCC-PBP by passage over a PD-10 desalting column (all chromatography materials were from Amersham Biosciences). Unlabeled phosphate-binding protein and Pi-insensitive MDCC-PBP were removed by passage over a Q-Sepharose column and a Mono-Q column, respectively (45Brune M. Hunter J.L. Howell S.A. Martin S.R. Hazlett T.L. Corrie J.E.T. Webb M.R. Biochemistry. 1998; 37: 10370-10380Crossref PubMed Scopus (172) Google Scholar). The final preparation had a 12-15-fold increase in fluorescence at 465 nm on binding Pi, indicating that the protein was largely Pi-free.Removal of contaminating inorganic phosphate from buffers, the final enzyme preparation, and the stopped-flow spectrometer required a “Pi mop,” assembled as described by Nixon et al. (46Nixon A.E. Hunter J.L. Bonifacio G. Eccleston J.F. Webb M.R. Anal. Biochem. 1998; 265: 299-307Crossref PubMed Scopus (40) Google Scholar). The Pi mop sequesters Pi into the stable ribose 5-phosphate molecule through a coupled enzyme system. Bacterial purine nucleoside phosphorylase and 7-methylguanosine converts phosphate and ribose to ribose 1-phosphate, which is then modified by phosphodeoxyribomutase (PDRM) in the presence of MnCl2 and α-d-glucose 1,6-bisphosphate to form the stable ribose 5-phosphate.Purine nucleoside phosphorylase, 7-methylguanosine, and α-d-glucose 1,6-bisphosphate were purchased from Sigma. Purified PDRM was made by amplifying the E. coli PDRM gene from strain XL1 blue genomic DNA by PCR using primers 5′-ACTCCATGGAACGTGCATTTATTATGGTTCTGGACTCATTCGG-3′ and 5′-ATGCTCGAGTCAGAACATTTGCTTTGCCATATTCCATATCAG-3′. The primers included an NcoI site over the ATG start codon and an XhoI site down-stream of the native stop codon (the restriction sites are depicted by the underscored sequences). The open reading frame was verified by sequencing the entire insert, which was then ligated into the pHIS-Parallel1 vector (47Sheffield P.J. Garrard S.M. Derewenda Z.S. Protein Expression Purif. 1999; 15: 34-39Crossref PubMed Scopus (525) Google Scholar) and used for expression in BL21(DE3) cells (Invitrogen). The PDRM protein was purified from cell lysates via an amino-terminal His6 affinity tag.The kinetics of Pi release were followed in an Applied Photophysics SX.18MV-R stopped-flow spectrometer (Surrey, UK). The fluorescence change of MDCC-PBP was monitored at the excitation wavelength of 425 nm with a 455-nm emission cutoff filter. The syringe contents, listed in the figure legends, were essentially the same as for the rapid quench flow experiments except for the lack of radioactivity and the addition of 20 μm MDCC-PBP. The Pi binding response of MDCC-PBP fluorescence was calibrated using known Pi concentrations in the stopped flow at fixed photomultiplier voltage.Kinetic Analysis—The experimental data fitting program, Scientist version 2.0 (Micromath Research, Inc., St. Louis), was used to fit the experimental data by numerical integration of differential equations described in detail previously (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar). The calculated rate constants were evaluated based on the least squares regression value, R2, and model selection criteria) (see Ref. 48MicroMath_Research MicroMath Scientist: For Experimental Data Fitting/Microsoft Windows. MicroMath Research, St. Louis1995Google Scholar and Baylis Scanlon et al. (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar)) values from statistical analyses performed to determine the goodness-of-fit.General Methods and Materials—Except where [γ-32P]ATP was used, steady state ATPase activities were determined as described previously (37Al-Shawi M.K. Ketchum C.J. Nakamoto R.K. J. Biol. Chem. 1997; 272: 2300-2306Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) using the Taussky and Shorr colorimetric method (49Taussky H.H. Shorr E. J. Biol. Chem. 1953; 202: 675-685Abstract Full Text PDF PubMed Google Scholar). The nucleotide content of cross-linked and uncross-linked F1 was determined by an ion exchange high pressure liquid chromatography assay using a Titansphere TiO2 column (Alltech Assoc., Deerfield, IL) as described previously (18Baylis Scanlon J.A. Al-Shawi M.K. Le N.P. Nakamoto R.K. Biochemistry. 2007; 46: 8785-8797Crossref PubMed Scopus (29) Google Scholar). Protein concentrations were determined using the method of Lowry et al. (50Lowry O.H. Rosebrough N.J. Farr A.C. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and in most instances cross-checked by the Amido Black protein assay (51" @default.
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• W2013906099 title "A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis" @default.
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