A nuclear mutation conferring aurovertin resistance to yeast mitochondrial adenosine triphosphatase.

A single gene nuclear yeast mutant was isolated whose mitochondrial F1-ATPase was resistant to the specific F1 inhibitor aurovertin. The mutant enzyme was not cross-resistant to other F1 inhibitors. The binding of aurovertin to F1 and to the two largest F1 subunits (alpha and beta) was measured by enhancement of aurovertin fluorescence. Aurovertin bound to wild type F1-ATPase and to its monomeric beta subunit with about the same binding constant. It failed to bind to wild type alpha subunit or to either F1 or F1 subunits from the mutant. The aurovertin-resistant mutant thus contains an altered nuclear gene which specifies the structure of the beta subunit of F1.     

Binding of Mg2+ to the beta subunit or F1 of H+-ATPase from Escherichia coli

The bindings of Mg2+ to the F1 portion of Escherichia coli H+-ATPase and its isolated alpha and beta subunits were studied with 8-anilinonaphthalene-1-sulfonate (ANS). The fluorescence of ANS increased upon addition of F1 or its alpha subunit or beta subunit, as reported previously (M. Hirano, K. Takeda, H. Kanazawa, and M. Futai (1984) Biochemistry 23, 1652-1656). The fluorescence of ANS bound to F1 or its beta subunit increased significantly with further addition of Mg2+, whereas that of the alpha subunit increased only slightly. Ca2+ and Mn2+ had similar effects on the fluorescence of ANS with F1 and its beta subunit. The Mg2+-induced fluorescence enhancement (delta F) was high at an alkaline pH and was lowered by addition of ethylenediaminetetraacetic acid. Dicyclohexylcarbodiimide and azide had no effect on the delta F. Binding analysis showed that the concentration dependence of Mg2+ on the fluorescence enhancement of the beta subunit is similar to that of F1. These results suggest that both the beta subunit and F1 have binding sites for Mg2+ and that the delta F observed with F1 may be due to the binding of Mg2+ to the beta subunit.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

Mutational replacements of conserved amino acid residues in the beta subunit resulted in defective assembly of H+-translocating ATPase (F0F1) in Escherichia coli

Mutant genes for the beta subunit of H+-translocating ATPase (F0F1) were cloned from Escherichia coli strains isolated in this laboratory. Determination of their nucleotide sequence revealed four missense mutations (strain KF39, Glu-41----Lys; strain KF16 and KF42, Glu-185----Lys; strain KF48, Gly-223----Asp; KF26 and 4 other strains, Ser-292----Phe). Two nonsense mutants (strain KF40, Gln-361----end; strain KF20, Gln-397----end) were also identified. Glu-41, Glu-185, and Ser-292 are conserved in the amino acid sequences of the beta subunits so far studied, and Gly-223, Gln-361, and Gln-397 are conserved in beta subunits from bacteria and mitochondria, but not in those from chloroplasts. The amounts of F1 subunits in the membranes of these strains were studied by immunochemical assay and two-dimensional gel electrophoresis. In the mutants studied, the amounts of alpha and beta subunits in the membranes were 69-21 and 46-2%, respectively, of the amounts in wild-type membranes, the amount depending on the strain. No delta and epsilon subunits were detected in membranes of a missense mutant KF16, although reduced amounts of alpha and beta subunits could be detected, suggesting that the F1 portion may not be connected to F0 through the delta and epsilon subunits. The altered residues in missense mutants or missing domains in nonsense mutants may be important for the subunit-subunit interactions or assembly of the entire complex. Genetic experiments on introduction of suppressor tRNA into strains KF40 and KF20 suggested that F1 could be active even when residue 361 or 397 was replaced by a Ser, Leu, or Tyr residue.     

Aurovertin binds to the beta subunit of yeast mitochondrial ATPase.

Isolated beta subunit of ATPase (F1) from yeast mitochondria does not catalyze an ATPase reaction but still binds the specific F1 inhibitor aurovertin. Binding was measured by enhancement of aurovertin fluorescence; it was as tight as that to F1-ATPase. No binding was observed with F1 or with isolated beta subunit from a single-gene nuclear yeast mutant whose F1-ATPase was resistant to aurovertin.     

In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes

Using site-directed mutagenesis, Glu-190 or Glu-201 of the beta subunit of the F1-ATPase from the thermophilic bacterium PS3 were replaced with glutamine. It was possible to reconstitute complexes of the mutated beta subunits with alpha and gamma subunits, but the complexes did not have ATPase activity. It is concluded that carboxylic acid side chains of Glu-190 and Glu-201 of the beta subunit are essential for catalytic activity of F1-ATPase.     

Overproduction of truncated subunit a of H+-ATPase causes growth inhibition of Escherichia coli.

Genes (uncB) for wild-type and mutant a subunits of Escherichia coli H+-ATPase (F0F1) were cloned into recombinant plasmids. The subunits were expressed under the control of a weak promoter of the unc operon at 30 degrees C and strong promoters of lambda phage at 42 degrees C. At 30 degrees C, the wild type and a truncated (Glu-269----end) a subunit complemented the defect of the a subunit mutant KF24A (Trp-111----end), whereas the other mutant subunits (Trp-111----end, Trp-231----end, Gln-252----end, and a subunit with a deletion of residues 21 to 227) did not. Three mutant subunits (Trp-231----end, Gln-252----end, and Glu-269----end) and the wild-type a subunit caused growth inhibition associated with cell elongation, an uneven distribution of membrane proteins, and an altered septum structure when they were expressed at 42 degrees C. These phenomena were not observed with the other mutant subunits, suggesting that overproduction of the middle region (between residues 111 and 230) of the a subunit causes growth inhibition.     

Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies.

Aurovertins B and D inhibited the adenosine triphosphatase (ATPase) activity of soluble Escherichia coli coupling factor ATPase (BF1) isolated from wile-type E. coli K-12. Half inhibition was obtained with 2 microns aurovertin B and 0.9 microns aurovertin D. Aurovertins B and D had no inhibitory effect on BF1 isolated from the aurovertin-resistant E. coli mutant MA12. Acetylation or saponification of aurovertin D yielded a derivative which was devoid of inhibitory effect on BF1. Citreoviridin also inhibited wild-type BF1 but with much less efficiency (half inhibition at 60 microns) than aurovertin. Citreoviridin had no effect on the aurovertin-resistant BF1. The fluorescence intensity of aurovertins B and D was markedly enhanced upon addition to purified BF1. There was no enhancement of fluorescence when the aurovertins were added to BF1 isolated from the aurovertin-resistant mutant. The fluorescence of the aurovertin-BF1 complex was enhanced by adenosine 5'-diphosphate and by low concentrations of adenosine 5'-triphosphate. The adenosine 5'-diphosphate-enhanced fluorescence of the aurovertin-BF1 complex was quenched by high concentrations of adenosine 5'-triphosphate or by MG2+. Aurovertin bound selectively to the beta subunit of BF1 isolated from wile-type cells. By complementation assays in vitro, using a reconstituted system made of subunits isolated from wild-type and aurovertin-resistant BF1, it was shown that the altered peptide in aurovertin-resistant BF1 was the beta subunit.     

The glycine-rich sequence of the beta subunit of Escherichia coli H(+)-ATPase is important for activity

A short sequence motif rich in glycine residues, Gly-X-X-X-X-Gly-Lys-Thr/Ser, has been found in many nucleotide-binding proteins including the beta subunit of Escherichia coli H(+)-ATPase (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residues 149-156). The following mutations were introduced in this region of the cloned E. coli unc operon carried by a plasmid pBWU1: Ala-151----Pro or Val; insertion of a Gly residue between Lys-155 and Thr-156; and replacement of the region by the corresponding sequence of adenylate kinase (Gly-Gly-Pro-Gly-Ser-Gly-Lys-Gly-Thr) or p21 ras protein (ras) (Gly-Ala-Gly-Gly-Val-Gly-Lys-Ser). All F0F1 subunits were synthesized in the deletion strain of the unc operon-dependent on pBWU1 with mutations, and essentially the same amounts of H(+)-ATPase with these mutant beta subunits were found in membranes. The adenylate kinase and Gly insertion mutants showed no oxidative phosphorylation or ATPase activity, whereas the Pro-151 mutants had higher ATPase activity than the wild-type, and the Val-151 and ras mutants had significant activity. It is striking that the enzyme with the ras mutation (differing in three amino acids from the beta sequence) had about half the membrane ATPase activity of the wild-type. These results together with the simulated three-dimensional structures of the wild-type and mutant sequences suggest that in mutant beta subunits with no ATPase activity projection of Thr-156 residues was opposite to that in the wild-type, and that the size and direction of projection of residue 151 are important for the enzyme activity.     

Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.     

Coupling factor F1 ATPase with defective beta subunit from a mutant of Escherichia coli

The defective coupling factor F1 ATPase from a mutant strain (KF11) of Escherichia coli was purified to a practically homogeneous form. The final specific activity of Mg2+-ATPase was 6-9 units/mg protein, which is about 10-15 times lower than that of F1 ATPase from the wild-type strain. The mutant F1 had a ratio of Ca2+-ATPase to Mg2+-ATPase of about 3.5, whereas the wild-type F1 had ratio of about 0.8. The mutant F1 was more unstable than wild-type F1: on storage at -80 degrees C for 2 weeks, about 80% of its activity (dependent on Ca2+ or Mg2+) was lost, whereas none of the activity of the wild-type F1 was lost. The following results indicate that the mutation is in the beta subunit. (i) High Mg2+-ATPase activity (about 20 units/mg protein) was reconstituted when the beta subunit from wild type F1 was added to dissociated mutant F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. (ii) Low ATPase activity having the same ratio of Ca2+-ATPase to Mg2+-ATPase as the mutant F1 was reconstituted when a mixture of the beta subunit from the mutant F1 and the alpha and gamma subunits from wild-type F1 was dialyzed against the same buffer. (iii) Tryptic peptide analysis of the beta subunit of the mutant showed a difference in a single peptide compared with the wild-type strain.     

The binding of aurovertin to isolated beta subunit of F1 (mitochondrial ATPase). Stoicheiometry of beta subunit in F1.

1. Beef-heart mitochondrial ATPase (F1) is inactivated and dissociated by incubation with 0.85 M LiCl. ATP partly protects against inactivation. Three dissociation products could be identified after chromatography on diethylaminoethylcellulose: the delta subunit which is not adsorbed, the beta subunit which may be eluted from the column, and the alpha and gamma subunits which remain bound to the column. 2. Aurovertin binds to dissociated F1 with a fluorescence enhancement equal to about 30% that found with F1. Unlike intact F1 which shows two kinetically separated phases of fluorescence enhancement, only a fast phase is found with dissociated enzyme. 3. Fluorescence measurements at varying aurovertin and protein concentrations indicate that aurovertin binds to dissociated F1 in a simple 3-component reaction with dissociation constant 0.4 muM. There are two indistinguishable binding sites, calculated on the basis of the initial F1 concentration before dissociation. 4. The beta subunit was isolated from dissociated F1 by DEAE-cellulose chromatography. It has no ATPase activity but reacts with aurovertin with a fluorescence enhancement similar to that of dissociated F1. 5. The isolated beta subunit contains one aurovertin binding site with a dissociation constant of 0.56 muM. 6. It is concluded that F1 contains two beta subunits.     

Replacement of arginine 246 by histidine in the beta subunit of Escherichia coli H+-ATPase resulted in loss of multi-site ATPase activity

A mutant strain KF43 of Escherichia coli defective in the beta subunit of H+-translocating ATPase (F0F1) was examined. In this mutant, replacement of Arg246 by His was identified by DNA sequencing of the mutant gene and confirmed by tryptic peptide mapping. The mutant F1-ATPase was defective in multi-site hydrolysis of ATP but was active in uni-site hydrolysis. Studies on the kinetics of uni-site hydrolysis indicated that the k1 (rate of ATP binding) was similar to that of the wild-type, but the k-1 (rate of release of ATP) could not be measured. The mutant enzyme had a k3 (rate of release of inorganic phosphate) about 15-fold higher than that of the wild-type and showed 3 orders of magnitude lower promotion from uni- to multi-site catalysis. These results suggest that Arg246 or the region in its vicinity is important in multi-site hydrolysis of ATP and is also related to the binding of inorganic phosphate. Reconstitution experiments using isolated subunits suggested that hybrid enzymes (alpha beta gamma complexes) carrying both the mutant and wild-type beta subunits were inactive in multi-site hydrolysis of ATP, supporting the notion that three intact beta subunits are required for activity of the F1 molecule.     

Characterization of exchangeable and nonexchangeable bound adenine nucleotides in F1-ATPase from Escherichia coli

The total amount of bound exchangeable and nonexchangeable adenine nucleotides in Escherichia coli F1-ATPase (BF1) was determined; three exchangeable nucleotides were assessed by equilibrium dialysis in a [14C]ADP-supplemented medium. When BF1 was purified in a medium supplemented with ATP, a stoichiometry of nearly 6 mol of bound nucleotides/mol of enzyme was found; three of the bound nucleotides were ATP and the others ADP. When BF1 was filtered on Sephadex G-50 in a glycerol medium (Garrett, N.E., and Penefsky, H.S. (1975) J. Biol. Chem. 250, 6640-6647), bound ADP was rapidly released, in contrast to bound ATP which remained firmly attached to the enzyme. Upon incubation of BF1 with [14C]ADP, the bound ADP rather than the bound ATP was exchanged. Of the three [14C]ADPs which have bound to BF1 by exchange after equilibrium dialysis, one was readily lost by gel filtration on Sephadex G-50; the loss of bound [14C]ADP was markedly reduced by incubation of BF1 with aurovertin, a specific ligand of the beta subunit which is known to increase the affinity of the beta subunit for nucleotides (Issartel, J.-P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). Upon photoirradiation of BF1 with [alpha-32P]2-azido-ADP, only the beta subunit was labeled; concomitantly, bound ADP was released, but the content in bound ATP remained stable. These results suggest that specific sites located on the three beta subunits bind nucleotides in a reversible manner. Consequently, the tightly bound ATP of native BF1 would be located on the alpha subunits.     

Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis.

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

H(+)-ATPase gamma subunit of Escherichia coli. Role of the conserved carboxyl-terminal region

Cloned uncG genes (wild-type or in vitro mutagenized) for the Escherichia coli gamma subunit were introduced into the uncG mutant Gln-14----end), and the functions of the mutant subunits were studied. The F1's with Ala-283----end and Thr-277----end mutant gamma subunits had 63 and 14% of the wild-type ATPase activity, respectively, and mutants with these subunits showed reduced growth by oxidative phosphorylation, indicating that the 10 residues at the carboxyl terminus (286th residue) are important, but dispensable, for catalysis. On the other hand, F1 with a Gln-269----end gamma subunit was inactive. Replacement of conserved residues (Gln-269, Thr-273, or Glu-275) between Gln-269 and Leu-276 gave enzymes with significantly reduced ATPase activity (2-41% of that of the wild-type) and lower ATP-driven proton conduction. Thus these residues are required for the normal catalytic activity of F1, although they are not absolutely essential. Membranes with amino acid replacements (Thr-277----end, Gln-269----Leu, or Glu-275----Lys) and the frameshift mutation (downstream of Thr-277) had about 15% of the wild-type ATPase activity, but showed different degrees of ATP-dependent H+ translocation and growth yield by oxidative phosphorylation, suggesting that the gamma subunit, especially its carboxyl-terminal region, functions in coupling between catalysis and H+ translocation.     

Evidence for a nucleotide binding site on the isolated beta subunit from Escherichia coli F1-ATPase. Interaction between nucleotide and aurovertin D binding sites

The nucleotide binding capacity and affinity of the isolated beta subunit from Escherichia coli F1-ATPase have been studied with radiolabeled ADP and ATP by an equilibrium dialysis technique. Each mole of beta subunit in the presence of EDTA bound 1 mol of ADP or ATP with Kd values of 25 microM and 50-100 microM, respectively. At a saturating concentration, aurovertin enhanced the affinity of ADP or ATP for the isolated beta subunit by 3-6-fold. The Kd values for the binding of ADP or ATP were also assessed through the enhancing effect of ADP on [14C]aurovertin binding (Issartel, J.-P., Klein, G., Satre, M., & Vignais, P.V. (1983) Biochemistry 22, 3485-3492); the Kd values determined by this approach were several times lower than in the absence of aurovertin, in agreement with results obtained by direct titration with radiolabeled ADP or ATP.     

Three of the six possible intersubunit stabilizing interactions involving Glu-239 are sufficient for restoration of the homotropic and heterotropic properties of Escherichia coli aspartate transcarbamoylase

A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.     

Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli.

The effects of amino acid substitutions in the carboxyl terminal region of the H(+)-ATPase a subunit (271 amino acid residues) of Escherichia coli were studied using a defined expression system for uncB genes coded by recombinant plasmids. The a subunits with the mutations, Tyr-263----end, Trp-231----end, Glu-219----Gln, and Arg-210----Lys (or Gln) were fully defective in ATP-dependent proton translocation, and those with Gln-252----Glu (or Leu), His-245----Glu, Pro-230----Leu, and Glu-219----His were partially defective. On the other hand, the phenotypes of the Glu-269----end, Ser-265----Ala (or end), and Tyr-263----Phe mutants were essentially similar to that of the wild-type. These results suggested that seven amino acid residues between Ser-265 and the carboxyl terminus were not required for the functional proton pathway but that all the other residues except Arg-210, Glu-219, and His-245 were required for maintaining the correct conformation of the proton pathway. The results were consistent with a report that Arg-210 is directly involved in proton translocation.