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)

The Arg-41 of the c-subunit of the F0F1-ATPase of Escherichia coli has been changed by site-directed mutagenesis to Glu, Leu or Lys. None of the mutants can carry out oxidative phosphorylation. No detectable F1-ATPase activity is found on the membranes and only small amounts in the cytoplasm. Two-dimensional gel electrophoresis shows that in all three mutant strains the assembly of the F0F1-ATPase has been affected. When plasmids carrying the mutant genes, together with other normal unc genes, were inserted into strains each carrying a mutation in one of the unc genes other than uncE their capacity for oxidative phosphorylation was reduced or eliminated, the effect being most pronounced with the uncG and uncC mutants and least pronounced with the plasmid giving the Arg-->Lys substitution. The c-subunit is a multimer in the ATP synthase complex and it appears that a mixture of normal and mutant gene products allows assembly of a functional complex.
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PMID:The role of arginine in the conserved polar loop of the c-subunit of the Escherichia coli H(+)-ATPase. 844 8

Mutation of the alpha subunit of the Escherichia coli F1-ATPase to convert Arg-376 to a Cys (alpha R376C) lowers multisite ATPase activity 400-1,000-fold while affecting unisite catalysis only around 6-fold, suggesting that the mutation is in a region important for transmission of conformational changes between catalytic sites (Soga, S., Noumi, T., Takeyama, M., Maeda, M., and Futai, M. (1989) Arch. Biochem. Biophys. 268, 643-648; this study). To learn more of the structural features of the segment of the alpha subunit around Arg-376, mutant enzyme with a Cys at this position was modified with several maleimides. N-[14C]Ethylmaleimide reacted rapidly with this Cys in one of the three alpha subunits/F1 (2,500 M-1 s-1); more slowly with a second alpha subunit (390 M-1 s-1); and the same Cys in the third copy of the alpha subunit was completely unreactive to the reagent, indicating asymmetry of alpha subunits in the ECF1 complex. The photoactivatable cross-linker N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide++ +, when reacted via its maleimide to alpha Cys-376 of the mutant, covalently linked alpha to beta subunits upon photolysis, indicating that Cys-376 of alpha is close to an interface between the alpha and beta subunits. The EDTA-induced exchangeable noncatalytic site could be filled by TNP-ATP in both wild type and alpha R376C mutant ECF1. Occupancy of this site in the alpha R376C mutant altered the rate of reaction of the second-fastest reacting Cys-376 from 390 M-1 s-1 to below 130 M-1 s-1, suggesting that the two sites are on the same alpha subunit. TNP-ATP in the EDTA-induced exchangeable noncatalytic site was quenched by reacting Cys-376 with 4-maleimido-(2,2,6,6-tetramethylpiperidine-N(oxyl), indicating that the region around Cys-376, which is involved in transmission of conformational changes between alpha and beta, and noncatalytic sites are maximally 10-12 A from each other.
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PMID:The cysteine introduced into the alpha subunit of the Escherichia coli F1-ATPase by the mutation alpha R376C is near the alpha-beta subunit interface and close to a noncatalytic nucleotide binding site. 846 30

Mitochondria were prepared from three lymphoblast cell lines from patients with high percentage copy numbers of the human mtDNA 8993 mutation and compared to those prepared from related and non-related control cell lines. Rates of ATP synthesis with pyruvate/malate, succinate/rotenone, ascorbate/N'N'N'N' tetramethyl phenylene diamine were reduced to 67%, 58% and 54% of the control rates, respectively. The backward reaction measured as oligomycin sensitive ATPase was reduced to an average of 42% of that in controls. This mutation which changes a conserved leucine to an arginine in the putative membrane proton channel of mitochondrial ATPase effectively reduces the overall rate of oxidative phosphorylation.
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PMID:The mitochondrial DNA mutation at 8993 associated with NARP slows the rate of ATP synthesis in isolated lymphoblast mitochondria. 847 14

The gene encoding the epsilon subunit (atpE) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia coli. The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain epsilon that is as biologically active as epsilon purified from chloroplast-coupling factor 1 (CF1). Recombinant epsilon folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 deficient in epsilon and restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in epsilon. Site-directed mutagenesis was used to generate truncations and single amino acid substitutions in the primary structure of epsilon. In the five mutants tested, alterations that weaken ATPase inhibition by recombinant epsilon affect its ability to restore proton impermeability to a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeability. As in the case of E. coli, it appears that N-terminal truncations of the epsilon subunit have more profound effects than C-terminal deletions on the function of epsilon. Recombinant epsilon with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the epsilon of spinach and the epsilon of Pisum sativum, inhibits ATPase activity with a reduced potency similar to that of purified pea epsilon. Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in epsilon-CF1 interactions.
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PMID:Molecular dissection of the epsilon subunit of the chloroplast ATP synthase of spinach. 853 97

The alpha and beta subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the alpha subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln. The resultant mutant alpha3beta3gamma complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant alpha3beta3gamma complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding side was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.
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PMID:An attempt to convert noncatalytic nucleotide binding site of F1-ATPase to the catalytic site: hydrolysis of tethered ATP by mutated alpha subunits in the enzyme. 860 64

The Atp11p protein of Saccharomyces cerevisiae is required for proper assembly of the F1 component of the mitochondrial ATP synthase. The mutant atp11 genes were cloned and sequenced from 12 yeast strains, which are respiratory-deficient due to a defect in Atp11p function. Four of the mutations mapped to the mitochondrial targeting domain (amino-terminal 39 amino acids) of Atp11p. All the genetic lesions found in the mature protein sequence were shown to be nonsense mutations. This result is consistent with the idea that Atp11p activity is provided, principally, by the overall structure of a functional domain, and not by specific amino acid residues in a localized active site. Amino-terminal (Edman) sequence analysis of fragments derived from limited proteolysis of purified Atp11p, and in vivo functional characterization of deletion mutants, were employed to locate the position of the active region in the protein. Three domains, separated by proline-rich sequences, were identified in the mature protein. The active domain of Atp11p was mapped to the sequence between Phe-120 and Asn-174. The domains proximal (Glu-40 through Ser-109) and distal (Arg-183 through Asn-318) to the active region were found to be important for the protein stability inside mitochondria.
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PMID:Identification of functional domains in Atp11p. Protein required for assembly of the mitochondrial F1-ATPase in yeast. 861 60

The effect of metal chelators on protein import was investigated using isolated soybean mitochondria and soybean precursor proteins. Adding 1,10-phenanthroline, a metal chelator that can cross both mitochondrial membranes abolished import of both the alternative oxidase, and the F(A)d subunit of the ATP synthase, a matrix located protein. Other metal chelators such as EDTA, 1,7-phenanthroline and 4,7-phenanthroline, which cannot cross the mitochondrial membranes, had no effect on import. When processing, a known metal-dependent step inside mitochondria, was inhibited using a mutagenesis approach (changing a -2 arginine to a -2 glycine in the pre-piece of the precursor), so was import. Thus it would appear that in soybean, at least, translocation of proteins across the mitochondrial membrane, as well as processing, relies on a metal dependent step. Taken together, the data suggest the two processes may be directly connected in these mitochondria.
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PMID:Evidence for a link between translocation and processing during protein import into soybean mitochondria. 867 15

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the DeltauncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The DeltauncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit a, residues previously postulated to be essential in proton translocation. The aE219G and aE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the aGlu-219 residue cannot play a critical role in proton translocation. The aR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the aR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the DeltapH normally established by NADH oxidation with these membrane vesicles. aR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit a may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.
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PMID:On the role of Arg-210 and Glu-219 of subunit a in proton translocation by the Escherichia coli F0F1-ATP synthase. 940 80

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the FAd subunit of the ATP synthetase and the tobacco F1 beta subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bcl complex of the respiratory chain.
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PMID:A matrix-located processing peptidase of plant mitochondria. 948 72

A specific b subunit arginine, b(Arg-36) in Escherichia coli, displays evolutionary conservation among bacterial F1F0 ATP synthases. Site-directed mutagenesis was used to generate a collection of mutations affecting b(Arg-36). The phenotype differed depending upon the substitution, and the b(Arg-36-Glu) and b(Arg-36-Ile) substitutions virtually abolished enzyme function. Although the total amounts of F1F0 ATP synthase present in the membranes prepared from mutant strains were reduced, the primary effect of the b(Arg-36) substitutions was on the activities of the intact enzyme complexes. The most interesting result was that the b(Arg-36-Glu) substitution results in the uncoupling of a functional F0 from F1 ATP hydrolysis activity.
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PMID:Identification of an uncoupling mutation affecting the b subunit of F1F0 ATP synthase in Escherichia coli. 965 May 90


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