Novel Insights into the Chemical Mechanism of ATP Synthase
EVIDENCE THAT IN THE TRANSITION STATE THE gamma -PHOSPHATE OF ATP IS NEAR THE CONSERVED ALANINE WITHIN THE P-LOOP OF THE beta -SUBUNIT*

(Received for publication, January 27, 1997, and in revised form, April 14, 1997)

Young Hee Ko Dagger , Mario Bianchet §, L. Mario Amzel § and Peter L. Pedersen Dagger

From the Departments of Dagger  Biological Chemistry and § Biophysics and Biophysical Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2185

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 gamma -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. alpha , beta , gamma , delta , and epsilon ), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the beta  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 GX4GK(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 beta  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 beta  subunit within F1-ATPases and implicate alanine 158 as residing very near the gamma -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 GX4GK(T/S) sequence of many other nucleotide-binding proteins.


INTRODUCTION

Despite our extensive knowledge about the structure and function of the F1 moiety of ATP synthases (1-5), 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,1 a phosphate analog which in the presence of MgADP forms a transition state MgADP·Vi-myosin inhibitory complex (6-8). 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. 1A) (8, 9). These studies strongly implicated this serine as contacting directly the gamma -phosphate of ATP in the transition state, and were recently confirmed by x-ray structural analysis (10). The reaction pathway of F1-ATPase is believed to be quite similar to that of myosin-ATPase (11). In addition, within the catalytic sites of both enzymes resides the nucleotide binding consensus GX4GKT (P-loop) (12), which in the beta -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 gamma -phosphate of ATP in the transition state (10), 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 gamma -phosphate of ATP. Both goals were accomplished and provide novel insights into the chemical mechanism of ATP synthases.


Fig. 1. A, simplified scheme depicting how orthovanadate (Vi) interacts with nearby serine (or threonine) residues within proteins in an oxidation reaction activated by uv light. The dotted boxes represent a serine within a protein prior to the reaction (left), and the products of the reaction (middle and right). Following the uv and oxygen-dependent reactions, which result in peptide bond cleavage at two places in the serine residue, the amino group of the serine is covalently attached to the preceding amino acid in the protein, the side chain is oxidized to formic acid, and the alpha -carbon and carbonyl group are oxidized to the oxalyl moiety which remains at the N terminus of the subsequent amino acid. (See Ref. 27 for a more detailed description of the chemistry.) B, amino acid sequence of the rat liver F1-beta subunit deduced from cDNA showing the Walker A and B nucleotide binding consensus sequences (boxes). Threonine 163 which terminates the A consensus is depicted with an asterisk. The predicted catalytic base, glutamate 188, which lies between the A and B consensus regions, is underlined. The first N-terminal amino acid detected in the beta  subunit of isolated rat liver F1 is alanine. (See underlined region.)
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EXPERIMENTAL PROCEDURES

Materials

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 F1 beta -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.

Methods

Purification of Rat Liver F1-ATPase

The enzyme was purified by the procedure of Catterall and Pedersen (13) with the modification described by Pedersen et al. (14). The purified enzyme, in 250 mM KPi and 5.0 mM EDTA, 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 mM Tris-Cl, pH 7.5, or 50 mM MOPS, pH 8.0.

Preparation of Orthovanadate (Vi) Solution

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 Vi concentration 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.

Prior Treatment of F1 with Vi

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.

Assay for ATPase Activity

The spectrophotometric procedure was used in which ADP formed was coupled to the pyruvate kinase and lactic dehydrogenase reactions (13). 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 mM MgCl2, 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.

Assay for ADP Binding

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 mM Tris-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 (15, 16).


Fig. 4. Demonstration 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.
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SDS-PAGE

This was carried out in a Bio-Rad Mini-Protean dual slab cell in 15% acrylamide according to the method of Laemmli (17), or in cylindrical glass tubes in 5% acrylamide according to the method of Weber and Osborn (18). Where indicated, densitometric analysis of the Coomassie-stained bands was carried out using a Fujifilm Bas-1500 PhosphorImager and MacBas (V 2.31) software.

Western Blot Analysis

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 mM CAPS, 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 mM NaCl, 0.1% Tween 20, pH 7.5), incubated for 1 h at 23 °C with a rat liver F1-beta 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.

N-terminal Sequence Analysis

F1 beta -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-beta 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 (19) using an Applied Biosystems 475A Protein Sequencing System (20).

Determination of Protein

Protein was determined by the method of Lowry et al. (21) after first precipitating with 5% trichloroacetic acid.


RESULTS AND DISCUSSION

F1 Forms an Inhibitory Complex in the Presence of MgCl2, ADP, and Vi

To promote formation of an inhibitory MgADP·Vi-F1 transition state complex, several precautions were taken. First, the Vi stock 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. 2A 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, <1, 10, 20, and 20% under conditions described in the figure legend. In an experiment not presented, F1 prior incubated with MgCl2 alone, was not inhibited after 2 h. Finally, Fig. 2B shows that when F1 is prior incubated with 200 µM each of MgCl2, ADP, and Vi, but in the presence of light (320 nm) and atmospheric oxygen, an essentially identical inhibition profile is observed.


Fig. 2. Time dependent loss of F1-ATPase activity upon incubation of F1 with MgADP·Vi in 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 (bullet ), F1 + 0.2 mM ADP + 0.2 mM Vi (×), F1 + 0.2 mM MgCl2 + 0.2 mM ADP (triangle ), or F1 + 0.2 mM each of MgCl2, ADP, and Vi (black-diamond ). 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.
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Inhibition of F1-ATPase Activity by MgCl2 + ADP + Vi Is Reversible in the Absence of UV Light but Not in Its Presence

If formation of a MgADP·Vi-F1 transition state complex is responsible for the inhibition observed in the presence of MgCl2 + ADP + Vi, this state should be reversible. Results presented in Fig. 3A show that this is the case. Thus, following maximal inhibition of F1-ATPase activity in the presence of 200 µM each of MgCl2, ADP, and VI (Fig. 3A, left panel), an aliquot was removed and subjected to two dilution/wash cycles in the presence of 50 mM MOPS, 10% glycerol, pH 8.5. This resulted after two such cycles in the restoration of the original activity to a level near 90% (Fig. 3A, right panel). Significantly, reversal of ATPase activity could not be achieved (Fig. 3B, right panel) following inhibition of F1-ATPase activity under identical conditions but in the presence of uv light (Fig. 3B, left panel) and with longer incubation times. The reason for this becomes clear in the description of other experiments described below.


Fig. 3. Demonstration 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 (black-diamond ) 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.
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Vi Induces Inhibition of F1-ATPase Activity under Turnover Conditions

It is expected that if a MgADP·Vi-F1 transition state is formed in the presence of MgCl2, ADP, and Vi as implicated from the above studies, it would be formed also under turnover conditions, i.e. when ATP, the substrate for ATP hydrolysis is present. For this reason, F1 was incubated exactly as described above but with ATP replacing ADP in the prior incubation mixture. Thus, the final prior incubation mixture contained 200 µM each of MgCl2, ATP, and Vi. After 1 h under these turnover conditions, aliquots were removed and assayed for ATPase activity. Results presented in Fig. 4 show that F1-ATPase activity is inhibited about 50% in the absence of uv light and about 70% in its presence. Although after 1 h the degree of inhibition is not as great as that achieved when prior incubation is carried out with ADP rather than ATP in the prior incubation mixture, this is to be expected. Thus, under the latter conditions ATP must first undergo hydrolysis before ADP is available for formation of the MgADP·Vi-F1 complex and MgATP competes with ADP for binding to F1-ATPase active sites.

Polypeptide Chain Cleavage Occurs within the MgADP·Vi-F1 Complex in the Presence of UV Light

As indicated earlier, vanadate is photoreactive and has been shown in the case of the MgADP·Vi-myosin complex to modify an active site serine residue within contact distance of Vi and, in the presence of light and atmospheric oxygen, to induce peptide bond cleavage at this site (8, 9) (Fig. 1A). For this reason F1 was priorly incubated in the presence of 200 µM each of MgCl2, ADP, and Vi in the absence and presence of uv light exactly as described for Figs. 2 and 3 and then subjected to SDS-PAGE. When the resultant SDS-PAGE profiles (Fig. 5, A and B) of the two incubation mixtures (absence and presence of light) are compared, it is clear that only in the latter case has polypeptide bond cleavage occurred. Thus, in addition to Coomassie-stained bands corresponding to the known F1 subunits alpha , beta , gamma , delta , and epsilon , bands distinct from these subunits with apparent molecular masses of 17 and 34 kDa appear (Fig. 5B, lane 6). Polypeptide bond cleavage within the F1 molecule is highly specific, and is not observed in the absence of uv light under any condition tested (Fig. 5A), and is observed in the presence of uv light with MgCl2 + ADP + Vi (Fig. 5B, lane 6). Fig. 5C shows that the appearance of the 17- and 34-kDa peptide fragments increases with time as expected and levels off after about 2 h (lane 9). These findings are consistent with polypeptide bond cleavage within the nucleotide-binding consensus region (GGAGVGKT) of the 51.5-kDa beta -subunit, as this would give rise to two fragments with molecular masses near the experimentally determined values of the 17- and 34-kDa bands (Fig. 5C).


Fig. 5. SDS-PAGE gels demonstrating that a polypeptide chain is cleaved when the MgADP·Vi-F1 complex is exposed to uv light. A, absence of light (control). F1 was prior incubated for 2 h exactly as described in the legend to Fig. 2 either alone (lane 2) or in the presence of 0.2 mM Vi (lane 3), ADP + MgCl2 (lane 4), ADP + Vi (lane 5), MgCl2 + ADP + Vi (lane 6). Molecular weight markers are depicted in lane 1. B, presence of light. Conditions are identical to A except samples were subjected to uv light (see "Experimental Procedures") during the 2-h prior incubation period. C, time dependence of the polypeptide cleavage reaction in the presence of MgADP·Vi. Conditions are identical to those described in the legend to the Fig. 2 for ADP + MgCl2 + Vi. Prior incubation was carried for 17, 23, 33, 49, 61, 71, 83, and 114 min, respectively, for lanes 2-9; lane 10, control without prior incubation; lane 1, molecular weight standards.
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Polypeptide Chain Cleavage within the MgADP·Vi-F1 Complex Induced by UV Light Does Not Alter the Oligomeric State of F1 and Occurs Only within the beta -Subunit

Results of native PAGE experiments presented in Fig. 6A, lanes 1-5, show that, under conditions which result in cleavage of a polypeptide chain within the MgADP·Vi-F1 complex in the presence of uv light and atmospheric oxygen, the oligomeric state of the F1 molecule remains intact. In fact, the F1 molecule remains intact in uv light under all conditions tested (i.e. alone or with Vi, MgCl2 + ADP, ADP + Vi, or MgCl2 + ADP + Vi; Fig. 6A, lanes 1-5, respectively). Specifically, as it applies to the uv light and Vi-dependent cleavage of a polypeptide chain within the MgADP·Vi-F1 complex (Fig. 5B, lane 6), these results are consistent with a very localized reaction which is otherwise without deleterious effect on the remaining part of the F1 molecule.


Fig. 6. A, the oligomeric state of F1-ATPase is not altered by light activation of the MgADP·Vi-F1 complex. After prior incubation for 2 h of F1 alone or with the components indicated in legends to Figs. 2 and 3, in the presence of uv light, the incubation medium was subjected to native PAGE exactly as described under "Experimental Procedures." Lane 1, F1 alone; lane 2, F1 + Vi; lane 3, F1 + MgCl2 + ADP; lane 4, F1 + ADP + Vi; lane 5, F1 + MgCl2 + ADP + Vi. B, Western blot analysis of the cleavage products. Prior incubation was carried out for 2 h with F1 alone or with the components indicated in legends to Figs. 2 and 3, in the presence of uv light. Following SDS-PAGE, Western blot analysis using an anti-beta subunit antibody was carried out exactly as described under "Experimental Procedures." Lane 2, F1 alone; lane 3, F1 + Vi; lane 4, F1 + MgCl2 + ADP; lane 5, F1 + ADP + Vi; lane 6, F1 + MgCl2 + ADP + Vi. C and D, N-terminal sequence analysis of the 17- and 34-kDa cleavage products. N-terminal sequence analysis were carried out exactly as described under "Experimental Procedures" on the 17- and 34-kDa cleavage products derived from 3.5 µg of F1. E, stoichiometry of ADP binding to F1 with and without MgCl2 and orthovanadate. Binding assays were carried out at 25 °C exactly as indicated under "Experimental Procedures" in a 100-µl system containing 50 mM MOPS, pH 8.5, 10% glycerol (v/v), 150 µg of F1, and 200 µM of the indicated ligands. Values are reported as the mean ± S.D. The number of determination = n. F, quantification of the F1-beta subunit and the 17- and 34-kDa cleavage products. Quantification was carried using a PhosphorImager as described under "Experimental Procedures." Results presented are representative of more than five different experiments.
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Identification of the beta  or "catalytic" subunit within F1 as the source of the 17- and 34-kDa bands was derived from two separate experiments. In the first, SDS-PAGE gels of the uv light-treated MgADPVi·F1 complex, after transfer to PVDF membranes, were probed with a polyclonal, antibody raised against the synthetic peptide KIGLFGGAGVGKCT, containing the GX4GKT consensus region of the rat liver beta  subunit. As shown in Fig. 6B, lane 6, only the intact beta  subunit and the 17-kDa band cross-react with the antibody. This is the expected result if polypeptide bond cleavage occurs within the nucleotide binding consensus region of the beta  subunit, as the epitope reactive with the antibody would be largely retained at the C terminus of the 17-kDa fragment, but missing from the 34-kDa fragment. In the second experiment, N-terminal sequence analysis (Fig. 6C) which identified the 7-amino acid stretch APKAGTA confirmed the beta  subunit (Fig. 1B) as the origin of the 17-kDa fragment. (The first six amino acids at the N terminus of the rat liver beta  subunit predicted from c-DNA cloning (Fig. 1B) are not present in the isolated protein.) N-terminal sequence analysis of the 34-kDa fragment also proved possible by carrying out the transfer from SDS-PAGE to PVDF membranes for 2 rather than 1 h, and at 25 °C rather than 4 °C (see "Experimental Procedures"). This modification in the transfer procedure was done to promote removal of an oxalyl group (HO-CO-CO-) predicted to be at the N terminus of the 34-kDa fragment following the Vi-dependent cleavage reaction (Fig. 1A). Significantly, the N-terminal sequence obtained, GVGKTVLIMELINN (Fig. 6D), not only confirmed the 34-kDa fragment as being derived from the beta  subunit, but placed the site of cleavage at alanine 158 within the GGAGVGKT consensus region.

The Ratio of the Number of beta  Subunits Cleaved/F1 to the Number of beta  Subunits Binding ADP Is Near 1

To determine the extent of involvement of the 3 beta  subunits of F1 in the formation of the MgADP·Vi-F1 transition state complex, and in the formation of the 17- and 34-kDa cleavage products, both ADP-binding studies and densitometric analysis of Coomassie-stained bands were carried out. Fig. 6E shows that under the conditions used in this study, rat liver F1 binds only about 1 mol of ADP/mol of F1 and that Vi and MgCl2 have little or no affect on this stoichiometric ratio. When MgCl2, ADP, and Vi are added together, each at a concentration of 200 µM to favor formation of the MgADP·Vi-F1 complex (Fig. 2A), the stoichiometry of ADP binding remains constant. Thus, formation of the transition state complex appears to be restricted predominantly to the involvement of 1 beta  subunit. This correlates well with the estimated number of beta  subunits involved in formation of the 17- and 34-kDa cleavage products when the transition state complex is formed in the presence of uv light. Here, a loss of 33% of the total F1 beta  subunit staining intensity results which is fully recovered by the sum of the staining intensities of the 17- and 34-kDa products (Fig. 6F).

Studies with Sodium Borohydride Provide Further Evidence That an Alanine Residue Is Oxidized when the MgADP·Vi-F1 Complex Is Treated with UV Light

Studies described above provide rather compelling evidence that, in the presence of atmospheric oxygen, uv light-induced cleavage of a single beta  subunit within the MgADP·Vi-F1 complex occurs at alanine 158. If the beta -methyl group of this alanine residue is oxidized, it is expected to proceed through a series of reactions resulting first in the formation of serine, then an aldehyde (Fig. 7A), and finally other intermediates before peptide bond cleavage finally occurs (see Fig. 1A and Refs. 22 and 27). Therefore, it should be possible, via reduction of the aldehyde, to demonstrate a uv light-dependent incorporation of tritium [3H] from 3H-labeled sodium borohydride into F1-ATPase following the enzyme's inactivation by MgADP·Vi. Results presented in Fig. 7B show that such an incorporation does occur in very significant amounts over control levels obtained in the absence of MgADP·Vi. Incorporation reaches a maximal level in about 7 min, and then declines as expected because the aldehyde is a transient intermediate in the overall oxidation process.


Fig. 7. UV light-dependent incorporation of tritium from NaB[3H]4 into F1-ATPase following the enzymes inactivation by MgADP·Vi. A, predicted steps in the oxidation by Vi and oxygen of the methyl side chain of alanine. Note: one of the intermediates is predicted to be an aldehyde which can be reduced by NaBH4. B, incorporation of [3H] from Na[3H]4 into F1-ATPase. F1 was prior incubated with 0.2 mM each of MgCl2, ADP, and Vi in a 1.0-ml system containing 50 mM MOPS, 10% glycerol, pH 8.5, at 25 °C for 30 min, and then treated with uv light as indicated under "Experimental Procedures." At the times indicated 100-µl aliquots were withdrawn and subjected to column centrifugation as described under "Experimental Procedures" to remove excess Vi, MgCl2, and ADP. The resultant eluate was treated with 90 µM NaB[3H]4 (25 µCi/ml) at 25 °C for 45 min. The excess NaB[3H]4 was removed by column centrifugation. The entire eluate was then subjected to liquid scintillation counting. The radioactivity of [3H] at the zero time point was subtracted from that obtained at each time point and the difference (Delta CPM) was plotted as a function of time. Similar results were obtained in two additional experiments.
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Implications for the Chemical Mechanism of ATP Hydrolysis by F1

Studies reported here strongly implicate alanine 158 in the third position of the GGAGVGKT consensus of the rat liver F1 beta -subunit as residing very near the gamma -phosphate group of ATP in the transition state. Significantly, alanine in this position is conserved in all F1-ATPases (23), and in Escherichia coli, F1 mutations in this position to valine or proline result, respectively, in a >90% loss or a 2-fold activation of catalytic activity (24). Moreover, the comparable "third position" residue, serine 181, in Dictyostelium 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 (10). 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. 8A, center panel) from the pre-ATP hydrolysis state (Fig. 8A, left panel) is considered to align the Cbeta atom of alanine in close proximity to the gamma -phosphorus atom of ATP. In the proposed trigonal bipyramidal transition state complex, the planar gamma -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 beta  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 beta  subunits in catalysis via gamma ,beta -subunit interactions (5), it is not unreasonable to suggest also that these interactions may help stabilize the transition state.


Fig. 8. Scheme proposed for the initial events in the Vi-dependent oxidation of alanine 158 within the nucleotide binding consensus region of F1-ATPase. A, scheme depicting the relative positions of the Cbeta atom of alanine 158 and the gamma -phosphorus atom of ATP in the pre-hydrolysis state (left panel), the transition state (center panel), and post-hydrolysis state (right panel). Distances in the left and right panels were obtained from the coordinates of the x-ray structure of bovine heart F1 (5) kindly provided by Dr. J. E. Walker. :B represents an unknown base, involved either in the abstraction of a proton from water (5) or in sterochemically orienting and polarizing the attacking water without net proton abstraction (28). The hatched line between the CB carbon of alanine 158 and the gamma -P group of ATP in the transition state indicates that they are very near one another, not that there is a direct chemical interaction, although this possibility cannot be excluded. B, comparison of the amino acid sequences of the Walker A motif within myosin (rabbit muscle), adenylate kinase (chicken muscle), and F1-ATPase (rat liver). See Refs. 8 and 26, respectively, for myosin and adenylate kinase and refer to Fig. 1B of this paper for F1-ATPase.
[View Larger Version of this Image (28K GIF file)]

The mechanistic role of the third position serine in the GX4GK(T/S) sequence in myosin (8, 9) involves a much greater capacity to form hydrogen bonds to the oxygen of the gamma -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 gamma -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. 24 and 25). 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 (26). 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. 8B), 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 gamma -phosphate of ATP in the transition state. Significantly, in a recent intriguing paper Senior and colleagues (28) 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 gamma -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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant CA10951 (to P. L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-955-3827; Fax: 410-614-1944; E-mail: ppederse{at}welchlink.welch.jhu.edu.
1   The 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.

ACKNOWLEDGEMENTS

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.


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