Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.14 (ATP synthase)
7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deletion mutations in the NH2- and COOH-terminal regions of the epsilon subunit of Escherichia coli ATP synthase were constructed making use of the AatII and HincII restriction enzyme sites. The resultant mutated epsilon species were analyzed for in vivo functionality and for recognition by anti-epsilon monoclonal antibodies. Deletion of residues Asp-7 through Gln-14 (epsilon delta D7-Q14) resulted in reduced ability to complement uncC mutants as determined by growth yields on limiting glucose medium and by formation of small colonies on plates with succinate as the source of carbon and energy. None of the other mutants was notably impaired. Upon induction to obtain overexpression, the NH2-terminal deletion mutants were expressed at levels comparable to the wild-type epsilon subunit, but the COOH-terminal deletion mutants were expressed less strongly, suggesting that residues in the latter region are important for protein stability. Monoclonal antibody epsilon-1, which cannot bind to epsilon when it is part of F1-ATPase, recognized the COOH-terminal deletions well, but the NH2-terminal deletions poorly. Additional epitope mapping using epsilon fusion proteins revealed that residues required for the epsilon-1 epitope extend to between Thr-77 and Arg-85. Monoclonal antibody epsilon-4, which can bind to epsilon when it is part of F1-ATPase, recognized the NH2-terminal deletions well, but hardly recognized the COOH-terminal deletions, indicating a role of residues located COOH-terminal to Ile-131 in recognition by this antibody. Epitope mapping using the fusion proteins revealed that the residues required by epsilon-4 begin in the region between Val-78 and Met-95. These results imply a two-domain structure of epsilon and orient the subunit within the enzyme.
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PMID:Orientation of the epsilon subunit in Escherichia coli ATP synthase. 768 93

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.
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PMID:The ATP synthase gamma subunit. Suppressor mutagenesis reveals three helical regions involved in energy coupling. 777 64

Most F1F0 type ATP synthases, including that in Escherichia coli, use H+ as the coupling ion for ATP synthesis. However, the structurally related F1F0 ATP synthase in Propionigenium modestum uses Na+ instead. The binding site for Na+ residues in the F0 sector of the P. modestum enzyme. We postulated that Na+ might interact with subunit c of F0. Subunit c of P. modestum and E. coli are reasonably homologous (19% identity) but show striking variations around the H(+)-translocating, dicyclohexylcarbodiimide-reactive carboxyl (Asp61 in E. coli). Several hydrophobic residues around Asp61 were replaced with polar residues according to the P. modestum sequence in the hope that the polar replacements might provide liganding groups for Na+. One mutant from 31 different mutation combinations did generate an active enzyme that binds Li+, the combination being V60A, D61E, A62S, and I63T. Li+ binding was detected by Li+ inhibition of ATP-driven H+ transport, Li+ inhibition of F1F0-ATPase activity, and Li+ inhibition of F0-mediated H+ transport. The Li+ effects were observed with membrane vesicles prepared from a delta nhaA, delta nhaB mutant background which lacks Na+/H+ antiporters, and with purified, reconstituted preparations of F0 prepared from this background strain. Li+ inhibition was observed at pH 8.5 but not at pH 7.0. H+ thus appears to compete with Li+ for the binding site. Li+ binding was abolished by replacement of Glu61 by Asp or Ser62 by Ala. The side chains at Ala60 and Thr63 may act in a supporting structural role by providing a more flexible conformation for the Li+ binding cavity. Thr63 does not appear to provide a liganding group since H+ transport in two other mutants, with Gly or Ala in place of Thr63, was also inhibited by Li+. We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding, and that the flexibility provided by residues X and Y will prove crucial to this structure.
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PMID:Changing the ion binding specificity of the Escherichia coli H(+)-transporting ATP synthase by directed mutagenesis of subunit c. 781 24

The mutation of serine-174 to phenylalanine that causes a defect in the Escherichia coli F1-ATPase beta-subunit is suppressed by further mutations; Gly-149 to Ser, Ala-295 to Thr, Ala-295 to Pro, or Leu-400 to Gln (Miki, J., Fujiwara, K., Tsuda, M., Tsuchiya, T. and Kanazawa, H. (1990) J. Biol. Chem. 265, 21567-21572). We analyzed the effects of these second site mutations and of a newly identified Asn-158 to Tyr mutation on the activities of the ATPase without the original Ser-174 to Phe mutation. The beta-subunit with each amino acid replacement was expressed in the mutant strain JP17, which does not have a beta-subunit. Cells transformed with the plasmid carrying Ala-295 to Pro mutation alone did not grow on minimal medium agar supplemented with succinate as the sole carbon source, and showed 3% of the wild-type ATPase activity, suggesting that this mutation caused structural alterations affecting the catalytic function of the enzyme. Conversely transformants with other mutations grew well and had higher ATPase activities, suggesting that these mutations did not cause extensive structural alterations. From the transformants with the plasmid carrying the Ala-295 to Pro mutation, seven revertants capable of cell growth on succinate plates were isolated and reversion mutations were identified at residues 140, 159, 166, 171, 172 and 184 of the beta-subunits. The results suggested that Ser-174 and Ala-295 do not necessarily interact directly, but that the regions including these suppression mutation sites close to Ser-174, and Ala-295 interact with each other for the proper functioning of the ATPase. The ternary structure of the region surrounded by the residues which were identified as the reversion mutation sites for Ser-174 to Phe and Ala-295 to Pro mutations is important for the catalytic function of this enzyme.
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PMID:Residues interacting with serine-174 and alanine-295 in the beta-subunit of Escherichia coli H(+)-ATP synthase: possible ternary structure of the center region of the subunit. 806 Oct 38

Nucleotide binding proteins, including ras, elongation factor Tu, adenylate kinase, and the mitochondrial F1-ATPase have a glycine-rich motif known as the P-loop or the Walker A sequence (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). The primary structural constraints have been determined in the P-loop located in the beta-subunit of the mitochondrial ATPase from yeast. The primary structural constraints were determined for 9 residues that form the P-loop, 190Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr-Val198. Each residue was tested individually for possible functional replacements while keeping the primary structure of the remainder of the molecule constant. This analysis indicates with greater than 95% confidence that Gly190,Gly195, and Lys196 are invariant and Thr197 can only be replaced with Ser. The most alterable residue is Gly191, where 10 replacements, even Phe, form a functional enzyme. The remaining positions allow some amino acid replacements while restricting others. The primary structural constraints of the P-loop of the mitochondrial F1 suggests that the three-dimensional structure of the P-loop is similar to that of ras.
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PMID:Primary structural constraints of P-loop of mitochondrial F1-ATPase from yeast. 814 26

Subunit c of the H(+)-transporting F1F0 ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha-helices and function as a key component of the H(+)-translocase of F0. We report here the initial results of a structural study of purified subunit c in a chloroform-methanol-water (4:4:1) solvent mixture using standard two-dimensional NMR techniques. The spin systems of 78 of the 79 amino acid side chains have been assigned to residue type, and 44 of these have been assigned to specific residues in the sequence. Stretches of alpha-helical secondary structure were observed for Asp7-ILe26 in the first proposed transmembrane helix, and for Arg50-Ile55 and Ala67-Val78 in the second proposed transmembrane helix. Nuclear Overhauser effects (NOEs) were observed between residues at both ends of the predicted transmembrane helices. The intensities of the NOEs between helix-1 and helix-2 were not diminished by mixing of 2H-subunit c with 1H-subunit c, and therefore the NOEs must be due to intramolecular, rather than intermolecular, interactions. Hence the purified protein must fold as a hairpin in this solvent system, just as it is thought to fold in the lipid bilayer of the membrane. In native F0, dicyclohexylcarbodiimide reacts specifically with Asp61 in the second transmembrane helix of subunit c, and the rate of this reaction is reduced by substitution of Ile28 by Thr on the first transmembrane helix. The I28T substitution is shown here to alter the chemical shifts of protons at and around Asp61. This observation provides a further indication that subunit c may fold in chloroform-methanol-water solvent much like it does in the membrane.
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PMID:Helical structure and folding of subunit c of F1F0 ATP synthase: 1H NMR resonance assignments and NOE analysis. 821 94

The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the alpha and beta subunits of the ATP synthase. Various mutations were introduced in the alpha Lys-175 and alpha Thr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169-176) of the Escherichia coli ATP synthase alpha subunit. Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme. The entire enzyme assembly was lost by alpha Lys-175-->Phe (or Trp) or alpha Thr-176-->Phe (or Tyr) mutation. Other mutants had similar (alpha His-175, alpha Ser-175, alpha Gly-175, alpha Ser-176, and alpha His-176 mutants) or lower (alpha Ala-176, alpha Cys-176, alpha Leu-176, and alpha Val-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the alpha Ser-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as alpha Gly-175 and alpha His-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the alpha Lys-175 and alpha Thr-176 residues are not absolutely essential for catalysis, but that they, or possibly the entire conserved sequence, are located in the key domain for the subunit-subunit interactions essential for enzyme stability and steady state activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The alpha subunit of ATP synthase (F0F1): the Lys-175 and Thr-176 residues in the conserved sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) are located in the domain required for stable subunit-subunit interaction. 826 95

Subunit c from the F1Fo ATP synthase of Escherichia coli folds in a hairpinlike structure of two alpha-helices in a solution of chloroform-methanol-H2O, and thus resembles the structure predicted for the folded protein in the membrane. The relevance of the structure in solution to the native structure was demonstrated. Asp61 in the second helical arm was shown to retain its unique reactivity with dicyclohexylcarbodiimide (DCCD) in chloroform-methanol-H2O solution. Further, the protein purified from the Ile28-->Thr DCCD-resistant mutant proved to be less reactive with DCCD in solution. This suggested that the protein folded with Ile28 of the first helical arm close to Asp61 in the second helical arm. Subunit c in wild-type E. coli membranes was specifically labeled with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified. DQF COSY spectra were recorded, and the distances between the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the 1H resonances. The paramagnetic contribution to T2 relaxation in the NCCD-labeled sample was calculated by an iterative computer-fitting method, where a control spectrum of a phenylhydrazine-reduced sample was broadened until the line shape of one-dimensional slices through each COSY cross-peak maximally mimicked the line shape of the paramagnetic sample. The distances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca. 12 A) to the derivatized Asp61 in helix-2. A model for the interaction of helices in the NCCD-modified protein was generated by restrained molecular mechanics and molecular dynamics using 25 distances of < 10-20 A derived from paramagnetic broadening in combination with 15 long-range nuclear Overhauser enhancement (NOE) restraints (2-5 A) for distances between helices and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein.
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PMID:Hairpin folding of subunit c of F1Fo ATP synthase: 1H distance measurements to nitroxide-derivatized aspartyl-61. 829 94

The gamma subunit mutations, gamma Met-23-->Lys or Arg, in the Escherichia coli ATP synthase were previously reported to cause dramatically inefficient energy coupling between ATPase catalysis and H+ translocation (Shin, K., Nakamoto, R.K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). In this paper, we report that second-site mutations in the gamma subunit can suppress the effects of gamma Met-23-->Lys. By screening randomly mutagenized uncG (gamma Met-23-->Lys), eight mutations in the carboxyl-terminal region were identified; strains carrying gamma Arg-242-->Cys, gamma Gln-269-->Arg, gamma Ala-270-->Val, gamma Ile-272-->Thr, gamma Thr-273-->Ser, gamma Glu-278-->Gly, gamma Ile-279-->Thr, or gamma Val-280-->Ala in combination with gamma Met-23-->Lys were able to grow by oxidative phosphorylation. H+ pumping assayed in membranes prepared from double mutation strains demonstrated that efficient ATP-dependent H+ transport was restored. Interestingly, the single mutations, gamma Gln-269-->Arg or gamma Thr-273-->Ser, caused reduced growth by oxidative phosphorylation; however, when these mutations were in combination with gamma Met-23-->Lys, growth was substantially increased. Furthermore, strains carrying gamma Met-23-->Lys, gamma Gln-269-->Arg, or gamma Thr-273-->Ser as single mutations were temperature sensitive, whereas, strains with the double mutations, gamma Met-23-->Lys/gamma Gln-269-->Arg or gamma Met-23-->Lys/gamma Thr-273-->Ser, were thermally stable. Taken together, these results strongly suggest that gamma Met-23, gamma Arg-242, and the region between gamma Gln-269 to gamma Val-280 are close to each other and interact to mediate efficient energy coupling.
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PMID:The gamma subunit of the Escherichia coli ATP synthase. Mutations in the carboxyl-terminal region restore energy coupling to the amino-terminal mutant gamma Met-23-->Lys. 841 64

The beta Gly-149 residue is in a glycine-rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr; residues 149-156) of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit. Substitution of beta Gly-149 by Ser suppressed the effect of the beta Ser-174-->Phe mutation (Iwamoto, A., Omote, H., Hanada, H., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1991) J. Biol. Chem. 266, 16350-16355), suggesting that beta Gly-149 is located near beta Ser-174. In this study, we introduced different residues at position 149 and found that a single mutant beta Cys-149 was defective. The effect of beta Cys-149 mutation was suppressed by beta Gly-172-->Glu, beta Ser-174-->Phe, beta Glu-192-->Val, or beta Val-198-->Ala replacement. These results suggest that beta Gly-149, beta Gly-172, beta Ser-174, beta Glu-192, and beta Val-198 residues are located close together in the catalytic site. From these findings we propose a model of the catalytic site of the enzyme near the gamma phosphate moiety of ATP. F1 enzymes with the double mutations beta Cys-149/beta Glu-172, beta Cys-149/beta Phe-174, beta Cys-149/beta Val-192, and beta Cys-149/beta Ala-198 were less sensitive than wild-type F1 to dicyclohexylcarbodiimide and adenosine triphosphopyridoxal (an affinity analogue of ATP forming a Schiff base with the epsilon-amino group of beta Lys-155 or beta Lys-201), and became sensitive to N-ethylmaleimide in an ATP-protected manner. These results of inhibitor studies are consistent with the proposed model.
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PMID:Domains near ATP gamma phosphate in the catalytic site of H+-ATPase. Model proposed from mutagenesis and inhibitor studies. 842 92


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