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Novel Insights into the Chemical Mechanism of ATP Synthase

1997; Elsevier BV; Volume: 272; Issue: 30 Linguagem: Inglês

10.1074/jbc.272.30.18875

1083-351X

Young Hee Ko, Mario A. Bianchet, L. Mario Amzel, Peter L. Pedersen,

Adenosine and Purinergic Signaling

The chemical mechanism by which the F1 moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (Vi), a phosphate analog which has the potential of “locking” an ATPase, in its transition state by forming a MgADP·Vi complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the γ-phosphate of ATP. Upon incubating purified rat liver F1 with MgADP and Vi for 2 h to promote formation of a MgADP·Vi-F1 complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F1 (i.e. α, β, γ, δ, and ε), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the β subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F1-ATPases and the third residue within the nucleotide binding consensus GX 4GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP·Vi-F1 complex revealed about 1.0 mol/mol F1, while quantification of the peptide cleavage products revealed that no more than one β subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [3H] from NaB[3H]4 incorporates into MgADP·Vi-F1 complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single β subunit within F1-ATPases and implicate alanine 158 as residing very near the γ-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX 4GK(T/S) sequence of many other nucleotide-binding proteins. The chemical mechanism by which the F1 moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (Vi), a phosphate analog which has the potential of “locking” an ATPase, in its transition state by forming a MgADP·Vi complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the γ-phosphate of ATP. Upon incubating purified rat liver F1 with MgADP and Vi for 2 h to promote formation of a MgADP·Vi-F1 complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F1 (i.e. α, β, γ, δ, and ε), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the β subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F1-ATPases and the third residue within the nucleotide binding consensus GX 4GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP·Vi-F1 complex revealed about 1.0 mol/mol F1, while quantification of the peptide cleavage products revealed that no more than one β subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [3H] from NaB[3H]4 incorporates into MgADP·Vi-F1 complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single β subunit within F1-ATPases and implicate alanine 158 as residing very near the γ-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX 4GK(T/S) sequence of many other nucleotide-binding proteins. Despite our extensive knowledge about the structure and function of the F1 moiety of ATP synthases (1Capaldi R.A. Aggeler R. Turina P. Wilkins S. Trends Biochem. Sci. 1994; 12: 186-189Google Scholar, 2Pedersen P.L. Amzel L.M. J. Biol. Chem. 1993; 268: 9937-9940Abstract Full Text PDF PubMed Google Scholar, 3Senior A.E. Annu. Rev. Biophys. Biophys. Chem. 1990; 19: 7-41Crossref PubMed Scopus (329) Google Scholar, 4Bianchet M. Ysern X. Hullihen J. Pedersen P.L. Amzel L.M. J. Biol. Chem. 1991; 266: 21197-21201Abstract Full Text PDF PubMed Google Scholar, 5Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2763) Google Scholar), sufficient information is not available to write a chemical mechanism by which ATP is hydrolyzed and synthesized. In contrast, myosin-ATPase has been successfully studied using orthovanadate, Vi, 1The abbreviations used are: Vi, orthovanadate; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; PVDF, polyvinylidene difluoride; uv, ultraviolet. a phosphate analog which in the presence of MgADP forms a transition state MgADP·Vi-myosin inhibitory complex (6Goodno C.C. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2620-2624Crossref PubMed Scopus (279) Google Scholar, 7Goodno C.C. Proc. Natl. Acad. Sci. U. S. A. 1979; 79: 21-25Crossref Scopus (109) Google Scholar, 8Cremo C.R. Grammer J.C. Yount R.G. J. Biol. Chem. 1989; 264: 6608-6611Abstract Full Text PDF PubMed Google Scholar). Irradiation of this complex with uv light results in the modification of the single serine within the nucleotide binding consensus GESGAGKT followed by peptide bond cleavage at this site (Fig. 1 A) (8Cremo C.R. Grammer J.C. Yount R.G. J. Biol. Chem. 1989; 264: 6608-6611Abstract Full Text PDF PubMed Google Scholar,9Cremo C.R. Long G.T. Grammer J.C. Biochemistry. 1990; 29: 7982-7990Crossref PubMed Scopus (42) Google Scholar). These studies strongly implicated this serine as contacting directly the γ-phosphate of ATP in the transition state, and were recently confirmed by x-ray structural analysis (10Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (515) Google Scholar). The reaction pathway of F1-ATPase is believed to be quite similar to that of myosin-ATPase (11Cross R.L. Grubmeyer C. Penefsky H.S. J. Biol. Chem. 1982; 257: 12101-12105Abstract Full Text PDF PubMed Google Scholar). In addition, within the catalytic sites of both enzymes resides the nucleotide binding consensus GX 4GKT (P-loop) (12Walker J.E. Saraste M. Runswick M.H. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4269) Google Scholar), which in the β-subunit of F1 (GGAGVGKT), contains alanine in place of the internal Vi-sensitive serine characteristic of the myosin consensus (GESGAGKT). As this serine in myosin is known to contact the γ-phosphate of ATP in the transition state (10Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (515) Google Scholar), it seemed reasonable to assume that in F1 a nearby serine residing outside the consensus region or the terminal threonine within the consensus region may serve this role in the transition state. Alternatively, the third position within the consensus region of F1, although containing an alanine, may play an important role in the transition state of F1 as does the serine in the same position in myosin. Studies described below were carried out both to define optimal conditions for trapping F1-ATPase in a MgADP·Vi-F1 inhibitory transition state complex, and to establish in this state the identity of an amino acid residue near the γ-phosphate of ATP. Both goals were accomplished and provide novel insights into the chemical mechanism of ATP synthases. Rats (Harlan Sprague-Dawley, white males) were obtained from Charles River Breeding Laboratories. ATP, ADP, MgCl2, MOPS, CAPS, sodium orthovanadate, phosphoenolpyruvate, pyruvate kinase, and lactic dehydrogenase were obtained from Sigma. SDS, acrylamide, and bisacrylamide were from Bio-Rad. Ammonium sulfate and potassium phosphate were from J. T. Baker Chemical Co. Tuberculin syringes and Sephadex G-50 used in nucleotide binding assays were from Becton-Dickinson Co. and Pharmacia Biotech Inc., respectively. PVDF membranes were obtained from Millipore and Western blot reagents from Amersham. A polyclonal antibody against the rat F1β-subunit was raised in rabbits using the synthetic peptide KIGLFGGAGVGKCT. [3H]ADP and NaB[3H]4 were from NEN Life Science Products. All other reagents were of the highest purity commercially available. The enzyme was purified by the procedure of Catterall and Pedersen (13Catterall W.A. Pedersen P.L. J. Biol. Chem. 1971; 246: 4987-4994Abstract Full Text PDF PubMed Google Scholar) with the modification described by Pedersen et al. (14Pedersen P.L. Hullihen J. Wehrle J.P. J. Biol. Chem. 1981; 256: 1362-1369Abstract Full Text PDF PubMed Google Scholar). The purified enzyme, in 250 mm KPi and 5.0 mmEDTA, was divided into 100-μl aliquots, lyophilized to dryness, and stored at −20 °C. Prior to use, aliquots (150–250 μg) of lyophilized F1 were dissolved at 25 °C in 100 μl of water and precipitated twice with 3 m ammonium sulfate, 5 mm EDTA, redissolving between precipitations in 200 mm K2SO4, 10 mmTris-Cl, pH 7.5, or 50 mm MOPS, pH 8.0. To minimize the presence of polymeric species the following protocol was followed: Na3VO4 powder was dissolved in water and the pH adjusted with HCl to pH 10 (orange color). The solution was boiled for 2 min at which time the solution became clear. The pH was readjusted to pH 10 and the previous boiling repeated 2 times. After the optical density was determined at 265 nm, the Viconcentration was calculated using the molar extinction coefficient of 2925 m−1 cm−1. The stock solutions used in this study were 155 mm and were covered with aluminum foil and stored until use at −80 °C. F1 (50 μg) was priorly incubated in a 100- or 200-μl system containing 50 mm MOPS, pH 8.5, 10% glycerol (v/v) and, where indicated, also Vi, Vi + ADP, Vi + ADP + MgCl2, Vi + ATP + MgCl2, or MgCl2, all at concentrations indicated in legends. Prior incubations were carried out at 25 °C for the indicated times. In experiments where photoactivation of vanadate was induced with uv light (320 nm), the incubation mixture in an open Eppendorf tube was placed under a 100 watt, long wavelength mercury spot lamp (BLAK-RAY (Model B 100A, 115 V, 2.5 amperes; San Gabriel, CA) at a distance of 7.8 cm. The time in the presence of the light source varied as indicated in the figure legends. The spectrophotometric procedure was used in which ADP formed was coupled to the pyruvate kinase and lactic dehydrogenase reactions (13Catterall W.A. Pedersen P.L. J. Biol. Chem. 1971; 246: 4987-4994Abstract Full Text PDF PubMed Google Scholar). The reaction mixture contained the following in a volume of 1 ml at pH 7.5 and 25 °C: 0.2 mm ATP, 65 mm Tris-Cl, 4.8 mmMgCl2, 2.5 mm KPi, 0.40 mm NADH, 0.60 mm phosphoenolpyruvic acid, 5 mm KCN, 1 unit of lactic dehydrogenase, 1 unit of pyruvate kinase, and 1.5 μg of F1. Binding assays were carried out at 25 °C by incubating F1, or fractions derived therefrom, for 20 min in a final volume of 100 μl, containing concentrations of F1, [3H]ADP, MgCl2, Vi, and buffer as indicated in the legend to Fig. 4. The entire reaction mixture was loaded onto a Sephadex G-50 “fine” column (1-cm3 tuberculin syringe with a filter at the bottom), which had been pre-equilibrated with 50 mmTris-Cl, pH 7.6, and priorly centrifuged for 1.5 min at 2,500 rpm in an IEC model HN-SII clinical centrifuge. Centrifugation of the reaction mixture was carried out for 1.5 min at 2,500 rpm to separate nucleotide bound to F1 from free nucleotide (15Williams N. Hullihen J.M. Pedersen P.L. Biochemistry. 1987; 26: 162-169Crossref PubMed Scopus (25) Google Scholar, 16Penefsky H.S. J. Biol. Chem. 1977; 252: 2891-2899Abstract Full Text PDF PubMed Google Scholar).Figure 3Demonstration that the inhibition of F1-ATPase activity by MgCl2 + ADP + Vi is reversible in the absence of uv light (A) and irreversible in its presence (B). Prior incubations were carried out with F1-ATPase in the absence () or presence (♦) of MgCl2, ADP, and Vi exactly as described in the legend to Fig. 2 and under “Experimental Procedures.” After inhibition of ATPase activity had reached a maximal level (left panels, see arrows), a 0.1-ml aliquot was withdrawn and diluted 6-fold in 50 mm MOPS, 10% glycerol, pH 8.5. The diluted solution was filtered through Amicon's Microcon 100 Filtration Unit at 25 °C by centrifugation at 500 × g for 15 min. The filtrate was discarded and the retentate was diluted to 0.1 ml and assayed for F1-ATPase activity as described under “Experimental Procedures” (right panels, Cycle 1). The dilution, washing, assay procedures were then repeated (right panels, Cycle 2). The entire experiment was repeated with essentially identical results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Time dependent loss of F1-ATPase activity upon incubation of F1 with MgADP·Viin the absence (A) and presence of uv light (B). Prior incubation was carried out as indicated under “Experimental Procedures” in a 0.1-ml system containing F1 alone (), and where indicated, F1 + 0.2 mm Vi(•), F1 + 0.2 mm ADP + 0.2 mmVi (×), F1 + 0.2 mmMgCl2 + 0.2 mm ADP (▵), or F1 + 0.2 mm each of MgCl2, ADP, and Vi(♦). At the indicated times, a 3-μl aliquot (1.5 μg F1) was withdrawn and assayed for ATPase activity exactly as described under “Experimental Procedures.” The results presented are representative of more than five different experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Demonstration that Vi induces inhibition of F1-ATPase activity under turnover conditions. Prior incubation was carried out at 25 °C with F1-ATPase in the absence or presence of 200 μm each of MgCl2, ATP, and Vi. After 1 h, 3-μl aliquots (1.5 mg F1) were withdrawn and assayed for ATPase activity as described under “Experimental Procedures.” Where indicated a control was also carried out under identical conditions but with ADP rather than ATP in the prior incubation mixture. Dark bars, absence of light;shaded bars, presence of light. The data presented are averages of duplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This was carried out in a Bio-Rad Mini-Protean dual slab cell in 15% acrylamide according to the method of Laemmli (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207509) Google Scholar), or in cylindrical glass tubes in 5% acrylamide according to the method of Weber and Osborn (18Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar). Where indicated, densitometric analysis of the Coomassie-stained bands was carried out using a Fujifilm Bas-1500 PhosphorImager and MacBas (V 2.31) software. After conducting SDS-PAGE, the proteins on the gel were transferred electrophoretically onto a PVDF membrane (1 h at 100 volts and 0.2 amp at 4 °C in 10 mmCAPS, 10% methanol transfer buffer, pH 11). The product was then blocked for 1 h with 2% bovine serum albumin plus 5% nonfat dry milk in PBS-T (80 mm Na2HPO4, 20 mm NaH2PO4, 100 mmNaCl, 0.1% Tween 20, pH 7.5), incubated for 1 h at 23 °C with a rat liver F1-β subunit polyclonal antibody (see “Experimental Procedures”), and then further incubated for 1 h at 23 °C with the secondary antibody (horseradish peroxidase-conjugated anti-mouse IgG). The immunoreactive bands were detected by the enhanced chemiluminescene (ECL) system of Amersham Life Sciences. F1 β-subunit peptide fragments were transferred from SDS-PAGE gels onto PVDF membranes by electroblotting. Transfer conditions were for 1 h in 10 mm CAPS buffer, 10% methanol, pH 11, at 4 °C in the case of the 17-kDa F1-β subunit fragment, and 2 h in the same buffer at 25 °C in the case of the 34-kDa fragment. Only under the latter conditions could an N-terminal sequence be obtained with the 34-kDa fragment. The peptides were then excised and subjected to N-terminal sequencing (19Edman P. Acta. Chem. Scand. 1950; 4: 283-293Crossref Google Scholar) using an Applied Biosystems 475A Protein Sequencing System (20Hunkapillar M.W. Hood L.E. Science. 1983; 219: 650-659Crossref PubMed Scopus (102) Google Scholar). Protein was determined by the method of Lowry et al. (21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) after first precipitating with 5% trichloroacetic acid. To promote formation of an inhibitory MgADP·Vi-F1 transition state complex, several precautions were taken. First, the Vistock solution was priorly treated exactly as described under “Experimental Procedures” and maintained at pH 10 in the dark at −80 °C until use to prevent formation of polymeric species. Second, the zwitterionic buffer MOPS was used in all experiments as it both stabilizes F1 and prevents or minimizes inhibition by MgCl2 and the product MgADP (or ADP). Third, a pH of 8.5 was used in all experiments, as this pH is near optimal for the hydrolysis of ATP catalyzed by rat liver F1. Finally, equimolar amounts of MgCl2, ADP, and Vi were used to promote formation of MgADP·Vi at the active site of F1. Fig. 2 A summarizes results of an experiment where F1 was priorly incubated in the presence of 200 μm each of MgCl2, ADP, and Vi for the indicated times and then assayed for ATPase activity as described under “Experimental Procedures.” Here it is clear that under these conditions, which are optimal for forming a MgADP·Vi-F1 transition state complex, F1-ATPase activity is markedly inhibited. Half-maximal inhibition is reached in about 45 min, and maximal inhibition (∼80%) is reached in about 2 h. In control experiments with F1 alone, F1 + Vi, F1 + ADP, and F1 + MgADP, inhibition at 2 h is, respectively, 90% loss or a 2-fold activation of catalytic activity (24Takeyama M. Ihara K. Moriyama Y. Noumi T. Ida K. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1990; 265: 21279-21284Abstract Full Text PDF PubMed Google Scholar). Moreover, the comparable “third position” residue, serine 181, inDictyostelium myosin subfragment 1, has been shown to be within contact distance (2.6 Å) of the Vi oxygen atoms in the x-ray structure of the MgADP·Vi-myosin S1 complex (10Smith C.A. Rayment I. Biochemistry. 1996; 35: 5404-5417Crossref PubMed Scopus (515) Google Scholar). With these facts, and the extensive data presented in this paper in mind, a tentative pathway for ATP hydrolysis at the active site of F1 is proposed. Here, formation of the transition state (Fig. 8 A, center panel) from the pre-ATP hydrolysis state (Fig. 8 A, left panel) is considered to align the Cβ atom of alanine in close proximity to the γ-phosphorus atom of ATP. In the proposed trigonal bipyramidal transition state complex, the planar γ-phosphorus atom, a water molecule, and a potential catalytic base, “B,” are considered to be sufficiently close to facilitate ATP hydrolysis. As only one of the three β subunits forms a transition state complex in the presence of MgADP·Vi (Fig. 6, E and F), and F1 is believed to function by involving sequential participation of the β subunits in catalysis via γ,β-subunit interactions (5Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2763) Google Scholar), it is not unreasonable to suggest also that these interactions may help stabilize the transition state. The mechanistic role of the third position serine in the GX 4GK(T/S) sequence in myosin (8Cremo C.R. Grammer J.C. Yount R.G. J. Biol. Chem. 1989; 264: 6608-6611Abstract Full Text PDF PubMed Google Scholar, 9Cremo C.R. Long G.T. Grammer J.C. Biochemistry. 1990; 29: 7982-7990Crossref PubMed Scopus (42) Google Scholar) involves a much greater capacity to form hydrogen bonds to the oxygen of the γ-phosphate of ATP than does the alanine in the same position in F1-ATPase. Therefore, the third position serine in myosin may play a different role in the mechanism of this enzyme than does the third position alanine in F1-ATPases. Nevertheless, in both cases, this third position residue may also be critical in determining the polarity, size, and/or rate of formation of the binding pocket in which the γ-phosphate group of ATP is contained in the transition state. Significantly, differences in these parameters among different ATP-dependent enzymes may help “set” the catalytic rate at a value most compatible with an enzyme's physiological role, while determining in part substrate specificity (see also Refs. 24Takeyama M. Ihara K. Moriyama Y. Noumi T. Ida K. Tomioka N. Itai A. Maeda M. Futai M. J. Biol. Chem. 1990; 265: 21279-21284Abstract Full Text PDF PubMed Google Scholar and 25Tagaya M. Yagami T. Noumi T. Futai M. Kishi F. Nakazawa A. Fukui T. J. Biol. Chem. 1989; 264: 990-995Abstract Full Text PDF PubMed Google Scholar). Along these lines, it is interesting to note that Vi in the presence of uv light and oxygen also cleaves adenylate kinase at the third position (proline 17) within the nucleotide binding consensus GGPGSGKGT (26Cremo C.R. Loo J.A. Edmonds C.G. Hatlelid K.M. Biochemistry. 1992; 31: 491-497Crossref PubMed Scopus (53) Google Scholar). Thus, in support of the view proposed here, three different enzymes, myosin, F1-ATPase, and adenylate kinase are all cleaved at the same third position despite the fact that the amino acid occupying this position is very different in all cases (Fig. 8 B), but conserved within its specific enzyme class. Finally, it should be noted that results presented here, which have focused on alanine 158 of rat liver F1, do not preclude other amino acids near the γ-phosphate of ATP in the transition state. Significantly, in a recent intriguing paper Senior and colleagues (28Lobau S. Weber J. Wilke-Mounts S. Senior A.E. J. Biol. Chem. 1997; 272: 3648-3656Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) have summarized the possible roles of three catalytic site residues in the E. coli F1-ATPase. One of these, lysine 155 (lysine 162 in rat liver F1) is considered to be involved in the major functional interaction with the γ-phosphate of MgATP in the substrate bound or “ground state,” but to undergo conformational repositioning during catalysis. Therefore, when taken together with the novel findings from studies reported here, it will be of considerable interest to visualize the precise location and orientation of lysine 162 and alanine 158 in the transition state when the x-ray structure of the MgADP·Vi-F1 complex is elucidated. We are grateful to Dr. Albert Mildvan with whom we had many discussions about this work, Joanne Hullihen for technical assistance, and Jackie Seidl for processing the manuscript for publication.