Molecular mechanisms of rotational catalysis in the F(0)F(1) ATP synthase.

Rotation of the F(0)F(1) ATP synthase gamma subunit drives each of the three catalytic sites through their reaction pathways. The enzyme completes three cycles and synthesizes or hydrolyzes three ATP for each 360 degrees rotation of the gamma subunit. Mutagenesis studies have yielded considerable information on the roles of interactions between the rotor gamma subunit and the catalytic beta subunits. Amino acid substitutions, such as replacement of the conserved gammaMet-23 by Lys, cause altered interactions between gamma and beta subunits that have dramatic effects on the transition state of the steady state ATP synthesis and hydrolysis reactions. The mutations also perturb transmission of specific conformational information between subunits which is important for efficient conversion of energy between rotation and catalysis, and render the coupling between catalysis and transport inefficient. Amino acid replacements in the transport domain also affect the steady state catalytic transition state indicating that rotation is involved in coupling to transport.

Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein.

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.

Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state.

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.

Rotational coupling in the F0F1 ATP synthase.

The F0F1 ATP synthase is a large multisubunit complex that couples translocation of protons down an electrochemical gradient to the synthesis of ATP. Recent advances in structural analyses have led to the demonstration that the enzyme utilizes a rotational catalytic mechanism. Kinetic and biochemical evidence is consistent with the expected equal participation of the three catalytic sites in the alpha 3 beta 3 hexamer, which operate in sequential, cooperative reaction pathways. The rotation of the core gamma subunit plays critical roles in establishing the conformation of the sites and the cooperative interactions. Mutational analyses have shown that the rotor subunits are responsible for coupling and in doing so transmit specific conformational information between transport and catalysis.

ATPase activity of Chinese hamster P-glycoprotein.

We have developed two defined experimental systems for biochemical investigation of P-glycoprotein, namely, plasma membranes highly enriched in Pgp, obtained from the CR1R12 Chinese hamster ovary cell line, and pure, reconstituted Pgp, obtained by solubilization of Pgp from CR1R12 plasma membranes, Reactive Red 120 chromatography, and reconstitution in liposomes. Studies of the ATPase catalytic mechanism by kinetic methods and covalent inactivation have been greatly facilitated by the availability of these experimental systems. The technique of vanadate trapping of nucleotide has been particularly useful. As a result of these studies, we now have explicit, testable, proposals for (1) the normal catalytic pathway of ATP hydrolysis, (2) a postulated alternating catalytic site cycle, and (3) coupling of ATP hydrolysis to drug transport. The experimental methods described here should prove valuable for future studies of Pgp and of ABC transporters in general.

Intergenic suppression of the gammaM23K uncoupling mutation in F0F1 ATP synthase by betaGlu-381 substitutions: the role of the beta380DELSEED386 segment in energy coupling.

We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, gammaM23K, caused increased energy of interaction between gamma- and beta-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawi, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F1-ATPase [Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628] gammaM23K is believed to form an ionized hydrogen bond with betaGlu-381 in the conserved beta380DELSEED386 segment. In this report, we further test the role of gamma-beta-subunit interactions by introducing a series of substitutions for betaGlu-381 and gammaArg-242, the residue which forms a hydrogen bond with betaGlu-381 in the wild-type enzyme. betaE381A, D, and Q were able to restore efficient coupling when co-expressed with gammaM23K. All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by gammaM23K. betaE381K by itself caused inefficient coupling, but opposite from the effect of gammaM23K, the transition state thermodynamic parameters were lower than wild-type. These results suggest that the betaE381K mutation perturbs the gamma-beta-subunit interaction and the local conformation of the beta380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis. betaE381A, L, K, and R, and gammaR242L and E mutations perturbed enzyme assembly and stability to varying degrees. These results provide functional evidence that the beta380DELSEED386 segment and its interactions with the gamma-subunit are involved in the mechanism of coupling.

Mechanism of energy coupling in the FOF1-ATP synthase: the uncoupling mutation, gammaM23K, disrupts the use of binding energy to drive catalysis.

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits which caused inefficient transmission of coupling information between transport and catalysis [Al-Shawi, M. K. , Ketchum, C. J., and Nakamoto, R. K. (1997) J. Biol. Chem. 272, 2300-2306]. We hypothesized that the gammaM23K mutation, because of its effect on coupling, should alter the fundamental reactions steps that are normally modulated by DeltamuH+ via the coupling mechanism. In this paper, we address this issue by studying the thermodynamics of individual catalytic steps through the use of energy profiles to gain information regarding enzyme mechanism and the effects of the mutation. Compared to wild-type enzyme, the gammaM23K F1 had significant differences of two partial reactions: the rate constant for Pi release was 49-fold faster and the rate constant for ATP release was 8.4-fold faster than wild-type. These rate constants were considered together with characteristics of a group of F1 ATPase mutant enzymes and were analyzed quantitatively by linear free energy relationships [Al-Shawi, M. K., Parsonage, D., and Senior, A. E., (1990) J. Biol. Chem. 265, 4402-4410]. We found that the gammaM23K mutation prevents the proper utilization of binding energy to drive catalysis and blocks the enzyme in a Pi release mode. This finding is consistent with the use of energy from DeltamuH+ for increasing the affinity for Pi so that the substrate binds in a catalytically competent manner for synthesis of ATP. These results support the notion that the communication of coupling information is transmitted through the gamma-beta interface near gammaMet23 and beta380DELSEED386 segment.

The Escherichia coli FOF1 gammaM23K uncoupling mutant has a higher K0.5 for Pi. Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway.

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1. The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes. Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.

Energy coupling, turnover, and stability of the F0F1 ATP synthase are dependent on the energy of interaction between gamma and beta subunits.

Replacement of the F0F1 ATP synthase gamma subunit Met-23 with Lys (gammaM23K) perturbs coupling efficiency between transport and catalysis (Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). We demonstrate here that the gammaM23K mutation causes altered interactions between subunits. Binding of delta or epsilon subunits stabilizes the alpha3beta3gamma complex, which becomes destabilized by the mutation. Significantly, the inhibition of F1 ATP hydrolysis by the epsilon subunit is no longer relieved when the gammaM23K mutant F1 is bound to F0. Steady state Arrhenius analysis reveals that the gammaM23K enzyme has increased activation energies for the catalytic transition state. These results suggest that the mutation causes the formation of additional bonds within the enzyme that must be broken in order to achieve the transition state. Based on the x-ray crystallographic structure of Abrahams et al. (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the additional bond is likely due to gammaM23K forming an ionized hydrogen bond with one of the betaGlu-381 residues. Two second site mutations, gammaQ269R and gammaR242C, suppress the effects of gammaM23K and decrease activation energies for the gammaM23K enzyme. We conclude that gammaM23K is an added function mutation that increases the energy of interaction between gamma and beta subunits. The additional interaction perturbs transmission of conformational information such that epsilon inhibition of ATPase activity is not relieved and coupling efficiency is lowered.

The catalytic cycle of P-glycoprotein.

P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro. It contains two nucleotide-binding domains, and its structure places it in the 'ABC transporter' family. We review recent evidence that both nucleotide-sites bind and hydrolyse Mg-ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high-energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

The ATP synthase gamma subunit. Suppressor mutagenesis reveals three helical regions involved in energy coupling.

A role in coupling proton transport to catalysis of ATP synthesis has been demonstrated for the Escherichia coli F0F1 ATP synthase gamma subunit. Previously, functional interactions between the terminal regions that were important for coupling were shown by finding several mutations in the carboxyl-terminal region of the gamma subunit (involving residues at positions 242 and 269-280) that restored efficient coupling to the mutation, gamma Met-23-->Lys (Nakamoto, R. K., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 867-872). In this study, we used suppressor mutagenesis to establish that the terminal regions can be separated into three interacting segments. Second-site mutations that cause pseudo reversion of the primary mutations, gamma Gln-269-->Glu or gamma Thr-273-->Val, map to an amino-terminal segment with changes at residues 18, 34, and 35, and to a segment near the carboxyl terminus with changes at residues 236, 238, 242, and 246. Each second-site mutation suppressed the effects of both gamma Gln-269-->Glu and gamma Thr-273-->Val, and restored efficient coupling to enzyme complexes containing either of the primary mutations. Mapping of these residues in the recently reported x-ray crystallographic structure of the F1 complex (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), reveals that the second-site mutations do not directly interact with gamma Gln-269 and gamma Thr-273 and that the effect of suppression occurs at a distance. We propose that the three gamma subunit segments defined by suppressor mutagenesis, residues gamma 18-35, gamma 236-246, and gamma 269-280, constitute a domain that is critical for both catalytic function and energy coupling.

Inhibition of the ATPase activity of P-glycoprotein by porphyrin photosensitization of multidrug-resistant cells in vitro.

The effectiveness of photodynamic therapy against P-glycoprotein ATPase activity in multidrug-resistant cells was studied. Chinese hamster ovary AUXB1 (drug-sensitive) and CR1R12 (multidrug-resistant) cell lines were compared with respect to uptake of 14C-polyhematoporphyrin and porphyrin photosensitization. Phototoxicity of Photofrin was similar in both cell lines, and no major differences in uptake or efflux of 14C-polyhematoporphyrin were observed. Porphyrin photosensitization in vitro of CR1R12 cells or isolated plasma membranes from these cells caused inhibition of P-glycoprotein ATPase activity. Application of porphyrin photosensitization at a sublethal level to CR1R12 cells resulted in a small but significant increase in adriamycin-induced cytotoxicity. The hydrophobic "picket-fence" porphyrin, meso-tetrakis-(o-propionamidophenyl)porphyrin, alpha,alpha,alpha,beta-isomer, was more inhibitory toward P-glycoprotein ATPase activity than the two less hydrophobic porphyrins tetraphenylporphine tetrasulfonate and Photofrin.

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells.

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.

Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein.

A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.

Covalent inhibitors of P-glycoprotein ATPase activity.

Verapamil-stimulated ATP hydrolysis by Chinese hamster P-glycoprotein in plasma membranes was shown to occur at a site(s) which is conformationally flexible and of relatively low affinity and specificity. Such properties distinguish P-glycoprotein from other transport ATPases. 8-Azido-ATP and 2-azido-ATP were excellent substrates, confirming that both analogs are suitable photoaffinity labels for investigating the catalytic site(s). Inactivation of ATPase activity occurred coincident with covalent incorporation of approximately two 8-azido-ATP/P-glycoprotein, with the incorporated analog distributed equally between N- and C-terminal halves of the molecule. N-Ethylmaleimide potently inactivated in an ATP-protected, dithiothreitol-irreversible manner, with maximal inactivation occurring coincident with incorporation of approximately two N-ethyl-maleimide/P-glycoprotein. The critical catalytic site sulfhydryls were shown to be located equally in N- and C-terminal halves of the molecule. Sulfhydryl-substituted purines also gave substantial inhibition of P-glycoprotein ATPase activity, which was dithiothreitol reversible. The data provide guidelines for beginning investigation of catalytic site architecture by protein chemistry approaches.

Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase.

The "homology A" ("glycine-rich" or "P-loop") consensus sequence occurs in the catalytic sites of F1F0 ATP synthase enzymes. The conserved lysine of this motif is beta-subunit Lys-155 in Escherichia coli F1. The role of this lysine in binding and catalysis at the high affinity ATP binding site was studied with the mutants beta K155Q and beta K155E by measuring the rates of ATP binding/release, ATP hydrolysis/synthesis, and Pi release as a function of pH varied from 5.5 to 9.5. In wild type, protonated beta Lys-155 appears to contribute significantly to high affinity binding of ATP, probably through hydrogen bonding to the gamma-phosphate. ATP hydrolysis and synthesis were impaired strongly in the mutants, and the reaction equilibrium constant, which was pH-independent in wild type, was highly pH-dependent in beta K155Q and beta K155E. Studies of steady-state ATPase turnover showed that positive catalytic cooperativity was virtually absent and the pH-dependent component of positive catalytic cooperativity was abolished or reversed in the mutants. The data demonstrate that residue beta K155 is a critical catalytic residue in F1 ATPase.

Characterization of the adenosine triphosphatase activity of Chinese hamster P-glycoprotein.

A multidrug-resistant Chinese hamster ovary cell line (CR1R12) was obtained which constitutively expresses P-glycoprotein, up to 32% by weight of plasma membrane protein. CR1R12 plasma membranes had high, drug-activated ATPase activity referable to P-glycoprotein. The specific ATPase activity in the presence of verapamil was calculated to be approximately 9 mumol/min/mg (identical to 21 s-1) at 37 degrees C, pH 7.4. KM ATP was 1.4 mM, and ADP and 5'-adenylyl imidodiphosphate were competitive inhibitors with Ki values 0.35 and 0.44 mM, respectively. 2'-dATP was a good substrate, GTP and ITP were real but poor substrates, and ADP and AMP were not hydrolyzed. Optimal pH for ATP hydrolysis was 7.3. MgATP was the preferred substrate, and CaATP was hydrolyzed very weakly. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) covalently labeled the P-glycoprotein, and incorporation of 1.1 mol of NBD-Cl/mol of P-glycoprotein gave 100% inactivation. ATP protected against NBD-Cl inactivation. N-Ethylmaleimide was a potent inhibitor in the absence of ATP, and in its presence significant protection from inhibition could be achieved. Vanadate and fluoroaluminate were also strong inhibitors. The plasma membranes from CR1R12 cells should provide material for purification and reconstitution of P-glycoprotein and for screening of potential "multidrug-reversal" reagents by enzymic assay.

Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit.

Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.

Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.

Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.