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 amino acid sequence of an intrinsic inhibitor of mitochondrial ATPase isolated from yeast was completed by using solid-phase sequencing and conventional procedures. The inhibitor was found to be composed of 63 amino acid residues, to lack tryptophan, cysteine, and tyrosine, and to have a molecular weight of about 7,383. The inhibitor was characterized as a basic protein with 16 basic and 13 acidic amino acid residues, and several clusters of basic residues were noted. Some comments are made on the hydrophobic amino acids and the presence of repeated sequences.
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PMID:Amino acid sequence of an intrinsic inhibitor of mitochondrial ATPase from yeast. 645 1

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.
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PMID:A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes. 761 7

The asymmetry of Escherichia coli F1-ATPase (ECF1) has been explored in chemical modification experiments involving two mutant enzyme preparations. One mutant contains a cysteine (Cys) at position 149 of the beta subunit, along with conversion of a Val to Ala at residue 198 to suppress the deleterious effect of the Cys for Gly at 149 mutation (mutant beta G149C:V198A). The second mutant has these mutations and also Cys residues at positions 381 of beta and 108 of the epsilon subunit (mutant beta G149C:V198A:E381C/epsilon S108C). On CuCl2 treatment of this second mutant, there is cross-linking of one copy of the beta subunit to gamma via the Cys at 381, a second to the epsilon subunit (between beta Cys381 and epsilon Cys108), while the third beta subunit in the ECF1 complex is mostly free (some cross-linking to delta); thereby distinguishing the three beta subunits as beta gamma, beta epsilon, and beta free, respectively. Both mutants have ATPase activities similar to wild-type enzyme. Under all nucleotide conditions, including with essentially nucleotide-free enzyme, the three different beta subunits were found to react differently with N-ethylmaleimide (NEM) which reacts with Cys149, dicyclohexyl carbodiimide (DCCD) which reacts with Glu192, and 7-chloro-4-nitrobenzofurazan (NbfCl) which reacts with Tyr297. Thus, beta gamma reacted with DCCD but not NEM or NbfCl; beta free was reactive with all three reagents; beta epsilon reacted with NEM, but was poorly reactive to DCCD or NbfCl. There was a strong nucleotide dependence of the reaction of Cys149 in beta epsilon (but not in beta free) with NEM, indicative of the important role that the epsilon subunit plays in functioning of the enzyme.
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PMID:Asymmetry of Escherichia coli F1-ATPase as a function of the interaction of alpha-beta subunit pairs with the gamma and epsilon subunits. 765 34

The function and location of residue His-38 of the epsilon subunit of the Escherichia coli F1-ATPase were investigated. His-38 was replaced by glutamine and cysteine through site-directed mutagenesis to produce epsilon H38Q and epsilon H38C, respectively. Both epsilon H38Q and epsilon H38C fulfilled epsilon function in vivo as determined by growth on nonfermentable carbon sources, growth yield on limiting glucose, and recovery of cells from energy starvation conditions. epsilon H38Q and epsilon H38C were purified and studied in vitro. Pure epsilon H38C reacted rapidly with Ellman's reagent, indicating a surface location of the introduced cysteine. epsilon H38C which had been reconstituted with epsilon-depleted F1-ATPase could be linked specifically to the gamma subunit using two different heterobifunctional sulfhydril-reactive/photoreactive crosslinking agents, indicating that residue 38 lies near gamma. The mutated epsilon subunits were unaltered in their ability to inhibit epsilon-depleted F1-ATPase in vitro, even after modification of epsilon H38C with the bulky reagents fluorescein maleimide and N-(1-anilinonaphthyl-4)maleimide. It seems unlikely, therefore, that residue His-38 of epsilon interacts directly with gamma. Both the epsilon H38Q and epsilon H38C mutations reduced the recognition of epsilon by monoclonal antibody epsilon-1, but recognition of epsilon H38C was not further reduced by reaction with fluorescein maleimide. These results imply that residue 38 is not directly part of the epsilon-1 epitope, but plays a role in its formation.
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PMID:Location of conserved residue histidine-38 of the epsilon subunit of Escherichia coli ATP synthase. 768 92

Binding of ADP to the beta polypeptide isolated from the catalytic F1 portion (CF1) of the chloroplast ATP synthase caused an increase of 10-20% in the steady state fluorescence intensity of fluorescent maleimides attached to the cysteine residue at position 63. Fluorescence lifetime distributions indicated that the beta polypeptide switched between two conformational states depending on the presence or absence of bound ADP. The fluorescence enhancement induced by ADP binding allowed a direct calculation of the dissociation constant for ADP of 0.7 microM. ATP did not cause a fluorescence enhancement but competed with ADP for binding to the same site. An apparent dissociation constant of 2 microM was obtained for ATP binding. Fluorescence resonance energy transfer experiments indicated that Cys63 is 42 A away from the nucleotide binding site on the beta polypeptide, confirming a previous measurement [(Colvert, K.K., Mills, D.A., Richter, M.L. (1992) Biochemistry 31, 3930-3935]. Frequency domain fluorescence anisotropy measurements indicated that the beta polypeptide has an irregular, elongated shape which is in good agreement with the conformation found in the crystal structure of the beef heart mitochondrial F1 enzyme [Abrahams, J.P., Leslie, A.G.W., Lutter, R., & Walker, J.E. (1994) Nature 370, 621-628]. The rotational correlation time did not change significantly upon ADP binding, indicating that ADP did not induce a large change in the overall shape of the beta polypeptide. The results show that the nucleotide binding domain and the N-terminal domain of the beta polypeptide communicate with each other over a significant distance via conformational changes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:ADP binding induces long-distance structural changes in the beta polypeptide of the chloroplast ATP synthase. 774 14

Using vacuolar membranes from Neurospora crassa, we observed that sulfite prevented the loss of vacuolar ATPase activity that otherwise occurred during 36 h at room temperature. Sulfite neither activated nor changed the kinetic behavior of the enzyme. Further, in the presence of sulfite, the vacuolar ATPase was not inhibited by nitrate. We tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promoting the formation of disulfide bonds. All reducing agents tested, dithionite, selenite, thiophosphate, dithiothreitol and glutathione, prevented the loss of ATPase activity. On the other hand, all oxidizing agents tested, bromate, iodate, arsenite, perchlorate, and hydrogen peroxide, were potent inhibitors of ATPase activity. The inhibitory effect of the oxidizing agents was specific for the vacuolar ATPase. The mitochondrial ATPase, assayed under identical conditions, was not inhibited by any of the oxidizing agents. Analysis of proteins with two-dimensional gel electrophoresis indicated that nitrate can promote the formation of disufide bonds between proteins in the vacuolar membrane. These data suggest a mechanism to explain why nitrate specifically inhibits vacuolar ATPases, and they support the proposal by Feng and Forgac (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13244-13230) that oxidation and reduction of critical cysteine residues may regulate the activity of vacuolar ATPases in vivo.
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PMID:The vacuolar ATPase: sulfite stabilization and the mechanism of nitrate inactivation. 782 84

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli. They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b', bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment d' (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b', bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1.OSCP.b', F1.OSCP.bI or F1.OSCP.bC were formed, but b', bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1.OSCP.b'.F6 and F1.OSCP.bI.F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1.OSCP.b'.F6.d, F1.OSCP.b'.F6.d' and F1.OSCP.b.F6.d were isolated. In a further series of reconstitution experiments, the binary complexes b'.OSCP and b'.d, the ternary complex b'.d'.F6, and the quaternary complex OSCP.b'.F6.d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1.stalk complex, F1.OSCP.b'.d.F6, each contained one copy per complex of subunits b (or b'), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1:1:1. The ratio of b to d in purified F0 was 1:1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.
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PMID:ATP synthase from bovine heart mitochondria. In vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence. 793

Residue Tyr-341 of the F1-ATPase beta subunit from a thermophilic Bacillus strain, PS3, was mutagenized to leucine, cysteine or alanine. Each of the mutated beta subunits was isolated and its affinity for ATP-Mg was examined by means of difference circular dichroism and differential titration calorimetry. The Kd values for ATP-Mg obtained were: beta Y341 (wild type), 0.015 mM; beta Y341L, 0.7 mM; beta Y341C and beta Y341A, > 3 mM. All the mutant beta subunits could be reconstituted into the alpha 3 beta 3 gamma complex with alpha and gamma subunits. The alpha 3 beta (mutant)3 gamma complexes hydrolyzed ATP with apparent Vmax values larger than that of the alpha 3 beta (WILD)3 gamma complex. The apparent Km values of the alpha 3 beta (mutant)3 gamma complexes increased in parallel with the Kd values for ATP-Mg of the isolated mutant beta subunits. These results indicate that residue beta Y341 is directly involved in the catalytic ATP-Mg binding and is a major Km-determining residue of F1-ATPase.
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PMID:Tyr-341 of the beta subunit is a major Km-determining residue of TF1-ATPase: parallel effect of its mutations on Kd(ATP) of the beta subunit and on Km(ATP) of the alpha 3 beta 3 gamma complex. 808 97

When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.
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PMID:In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond. 826 35

The delta subunit of the F1-ATPase from Escherichia coli contains 2 cysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled with sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys140 by maleimides such as N-ethylmaleimide or fluorescein maleimide resulted in a reconstitutively active enzyme that was indistinguishable from the native protein. Labeling of Cys64 with or without concomitant labeling of Cys140 resulted in a reconstitutively inactive enzyme. The ATPase activity of either form of the labeled enzyme was unaffected. However, labeling of Cys64 was accompanied by dissociation of the delta subunit from the enzyme. These observations suggest a role for the microenvironment of Cys64 in interactions of the delta subunit with other subunits in the enzyme. Two types of evidence support the conclusion that the 2 cysteine residues of the delta subunit are in close proximity. First, incorporation of pyrene maleimide into both delta cysteines led to excimer formation. Second, incubation of F1 with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in quantitative formation of a disulfide bond between Cys64 and Cys140, presumably via disulfide interchange. The enzyme containing the internally cross-linked delta subunit exhibited an undiminished ability to support proton pumping when reconstituted into F1-depleted membrane vesicles. The presence of 2 closely apposed cysteinyl residues in the delta subunit of the native enzyme places constraints on the type of structure that may be proposed for the subunit.
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PMID:Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase. 830 87


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